Antibody against human insulin-like growth factor

ABSTRACT

For the effective treatment of diseases such as cancer in which hIGF participates, there have been desired to be developed antibodies which strongly bind to both factors hIGF-I and hIGF-II and inhibit their functions and fragments of these antibodies. The present invention provides antibodies which have the ability to specifically bind to human IGF-I and IGF-II to thereby inhibit the functions of human IGF-I and IGF-II and have binding activity with a binding constant of 5×10 9  M −1  or more measured with a biosensor BIACORE. In addition, the present invention provides diagnostics, preventives and remedies for an hIGF-mediated disease and a disease showing pathological progressing due to abnormally promoted hIGF production, which use said antibodies.

TECHNICAL FIELD

The present invention relates to an antibody to insulin-like growthfactor (hereinafter referred to as IGF) and an antibody fragment derivedfrom the antibody. The present invention further relates to a DNA codingfor said antibody and antibody fragment. The present invention relatesto recombinant vector comprising said DNA and transformant obtained byintroducing said recombinant vector into a host cell. The presentinvention further relates to methods for producing said antibody andantibody fragment using said transformant, and diagnostic, preventiveand therapeutic uses of said antibody and antibody fragment.

BACKGROUND ART

IGF is a factor which takes a very important role in controllingproliferation, differentiation and cell death (apoptosis) of epithelialcells of the breast, prostate, lung, colon and the like organs, and itsaction is carried out via an IGF receptor (hereinafter referred to asIGF-IR) existing on the cell surface (Endocrine Reviews, 16, 3-34,1995). Also, it is known that a protein called IGF-binding protein(hereinafter referred to as IGFBP) is existing and regulating theactivity of IGF promotively or inhibitively (Endocrine Reviews, 16,3-34, 1995).

As the IGF, two types of IGF-I and IGF-II exist, and each of themcomprises a single chain polypeptide and has about 40% homology with aninsulin precursor proinsulin at the amino acid level (Advances in CancerResearch, 68, 183-223, 1996). As the IGF-R three types of insulinreceptor exist, IGF-I receptor (hereinafter referred to as IGF-IR) andIGF-II receptor (hereinafter referred to as IGF-IIR). Each of theinsulin receptor and IGF-IR belongs to the tyrosine kinase type receptorfamily and exists on the cell membrane as an α₂β₂ hetero tetramer, afterforming S—S bond of a 135 kDa α subunit and 95 kDa of β subunit formedfrom a single chain precursor as a result of its digestion with aprotease (Endocrine Reviews, 16, 143-163, 1995, Breast Cancer Research &Treatment, 47, 235-253, 1998). The insulin receptor and IGF-IR haveabout 60% homology, and insulin and IGF-IR, and IGF and insulinreceptor, binds to each other though weak and act (Journal of BiologicalChemistry, 263, 11486-11492, 1988, Journal of Biological Chemistry, 268,7393-7400, 1993). The existstance of a hybrid receptor comprising the αβsubunit of insulin receptor and the αβ subunit of IGF-IR has beenproved, and it is considered that the hybrid receptor has high bindingaffinity for IGF-I than for insulin and acts as IGF-IR, but its role inintravital is unclear (Endocrine Reviews, 16, 3-34, 1995, EndocrineReviews, 16, 143-163, 1995). The IGF-IIR has a single chain structure,and there are three ligand-binding regions in its extracellular region.One of the ligand-binding regions is an IGF-II-binding region, and theother two are regions which bind to mannose-6-phosphate-containingproteins [renin, proliferin, thyroglobulin, endogenous transforminggrowth factor-β (TGF-β) and the like] (Endocrine Reviews, 16, 3-34,1995). It has been reported that the endogenous TGF-β is activated byits binding to IGF-IIR (Breast Cancer Research & Treatment, 52, 175-184,1998, Hormone & Metabolic Research, 31, 242-246, 1999). The IGF-IIR doesnot have tyrosine kinase activity and binds to only IGF-II among IGF.Since IGF-II is degraded by its binding to IGF-IIR, it is consideredthat IGF-IIR acts as an antagonist of IGF-II (Breast Cancer Research &Treatment, 52, 175-184, 1998).

Ten types of IGFBP (IGFBP-1 to IGFBP-10) have so far been known, and sixtypes among them (IGFBP-1 to IGFBP-6) have high binding affinity for IGF(Proceeding of the National Academy of Sciences of the United States ofAmerica, 94, 12981-12986, 1997). IGFBP-1 to IGFBP-6 have a high homologyof 40 to 60% at the amino acid level. It has been revealed that IGFBPregulates the function of IGF by undergoing various post-translationmodifications such as degradation and phosphorylation and therebyexerting influences upon the transfer of IGF, inhibition of degradationand binding to receptor (International Journal of Biochemistry & CellBiology, 28, 619-637, 1996, Endocrine Reviews, 18, 801-831, 1997).IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-5 have a case of promoting theaction of IGF and a case of inhibiting the same, and the actions ofIGFBP-2, IGFBP-3 and IGFBP-5 upon IGF are regulated by the degradationof IGFBP, and the action of IGFBP-1 by the phosphorylation of IGFBP-1,respectively (Endocrine Reviews, 16, 3-34, 1995, International Journalof Biochemistry & Cell Biology, 28, 619-637, 1996, Endocrinology &Metabolism Clinics of North America, 25,591-614, 1996). In addition, thebinding affinity of IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-5 for IGF isreduced when they bind to a specific receptor existing on the cellmembrane. As a result, IGFBP and IGF are dissociated to form free IGF(Endocrine Reviews, 16, 3-34, 1995, International Journal ofBiochemistry & Cell Biology, 28, 619-637, 1996, Endocrinology &Metabolism Clinics of North America, 25, 591-614, 1996). On the otherhand, IGFBP-4 and IGFBP-6 have the activity to inhibit the action of IGF(Endocrine Reviews, 16, 3-34, 1995, Endocrinology & Metabolism Clinicsof North America, 25, 591-614, 1996). In intravital, 90% or more of theblood IGF binds to IGFBP-3 and an acid-labile subunit, and exists in theform of a high molecular weight complex of about 150 kDa, therebyinhibiting degradation of IGF and its drain into the extra vascularregion (Journal of Biological Chemistry, 264, 11843-11848, 1989).

Both of the IGF-I and IGF-II show strong promoting proliferationactivity to a large number of cancer cells (sarcoma, leukemia, prostatecancer, breast cancer, lung cancer, colon cancer, gastric cancer,esophageal cancer, hepatic cancer, pancreatic cancer, renal carcinoma,thyroid gland cancer, brain tumor, ovarian cancer, uterine cancer)(British Journal of Cancer 65, 311-320, 1992, Anticancer Research, 11,1591-1595, 1991, Annals of Internal Medicine, 122, 54-59, 1995,Oncology, 54, 502-507, 1997, Endocrinology, 137, 1764-1774, 1996,European Journal of Haematology, 62, 191-198, 1999), and over-expressionof IGF has been identified in a large number of cancer cells (BritishJournal of Cancer, 65,311-320, 1992). Also, it has been reported thatexpression amounts of IGF-II and IGF-IR are more those in highermetastatic cancer cells than those in lower metastatic cancer cells(International Journal of Cancer, 65, 812-820, 1996). It has beenrevealed that such functions of IGF occur mainly via IGF-IR(Endocrinology 136, 4298-4303, 1995, Oncogene, 28, 6071-6077, 1999), butIGF-II also acts via the insulin receptor in breast cancer cells(Oncogene 18, 2471-2479, 1999).

It has been reported that, in the case of transgenic mice whichover-express IGF-I in prostate epithelial cells, about 50% of themdevelop prostate cancer after about 6 months (Proceedings of theNational Academy of Science of the United States of America, 97,3455-3460, 2000). Also, it has been shown that expression of IGF-I andIGF-IR is increased by the acquirement of androgen-independentproliferation ability in a human prostate cancer cell transplantationmodel mice (Cancer Research, 61, 6276-6280, 2001).

IGF is also concerned in the proliferation of cancer cells by mutuallyreacting with other factors. It has been reported that the activity ofIGF-I is increased and expression of IGF-I and IGF-IR is induced byestrogen in breast cancer cells (Endocrinology, 136, 1296-1302, 1995,Journal of Biological Chemistry, 265, 21172-21178, 1990, Journal ofSteroid Biochemistry & Molecular Biology, 41, 537-540, 1992, BritishJournal of Cancer, 75, 251-257, 1997). In addition, it is known thatestrogen inhibits production of IGFBP, reduces expression of IGF-IIR,and increases expression of IGFBP degrading enzyme in breast cancercells (Biochemical & Biophysical Research Communications, 193, 467-473,1993, Molecular Endocrinology, 5, 815-822, 1991).

On the contrary, it has also been reported that IGF-I increasesexpression of estrogen receptor (Endocrinology, 127, 2679-2686, 1990,Journal of Cellular Biochemistry, 52, 196-205, 1993), and that IGF-I andIGF-II increase the activity of estrone sulfatase which hydrolysesestrone sulfate into estrone, in breast cancer cells (InternationalJournal of Molecular Medicine, 4, 175-178, 1999).

In addition, IGF cooperatively acts with an epithelial cell growthfactor (epidermal growth factor; hereinafter referred to as EGF). Incervical cancer cells, EGF increases expression of IGF-II, and IGFincreases the growth activity of EGF (Proceedings of the NationalAcademy of Sciences of the United States of America, 92, 11970-11974,1995, Cancer Research, 2, 56, 1761-1765, 1996). It is also known thatEGF increases the amount of free IGF by inhibiting expression of IGFBP-3and thereby has a synergistic effect on cell growth activity (CancerResearch, 54, 3160-3166, 1994).

It is known that the function of several factors having anti-cellproliferation activity is exerted by inhibition of the IGF promotingactivity to proliferation. The function of TGF-β and retinoic acid toinhibit proliferation of breast cancer cells is exerted by theinhibition of the IGF function as a result of the induction of IGFBP-3expression (Journal of Biological Chemistry, 270, 13589-13592, 1995,Cancer Research, 56, 1545-1550, 1996, Endocrinology, 136, 1219-1226,1995). In addition, vitamin D and its synthetic derivatives inhibit thefunction of IGF to promote proliferation of beast cancer cells andprostate cancer cells, and the action is based on the increase of IGFBPexpression and inhibition of IGF-IR and IGF-II expression (Journal ofthe National Cancer Institute, 89, 652-656, 1997, Journal of MolecularEndocrinology, 20, 157-162, 1998, Journal of Endocrinology, 154,495-504, 1997, International Journal of Oncology, 13, 137-143, 1998).

It has been reported that tumor suppressor gene products also haveinfluence upon the function of IGF. For example, in sarcoma cells andthe like, the wild type p53 protein induces IGFBP-3 expression andinhibits IGF-II and IGF-IR expression (Nature, 377, 646-649, 1995,Cancer Research, 56, 1367-1373, 1996, DNA & Cell Biology, 17, 125-131,1998, Proceedings of the National Academy of Sciences of the UnitedStates of America, 93, 8318-8323, 1996, Endocrinology, 139, 1101-1107,1998). In breast cancer cells, on the contrary, it is known that the p53protein is phosphorylated by the function of IGF-I and is transportedfrom the nucleus into cytoplasm, thereby losing the function of p53protein (International Journal of Cancer, 55, 453-458, 1993). Inaddition to these, it has been reported that it is inhibited IGF-IRexpression by a Wilms' tumor suppressor gene product WT1 (Journal ofBiological Chemistry, 269, 12577-12582, 1994, 140, 4713-4724, 1999), andit is inhibited a mammary-derived growth inhibitor (MDGI) expression byIGF-I (International Journal of Oncology, 13, 577-582, 1998).

Relationship between life style such as energy intake and oncogenesishas been drawing attention from old times, and it is now partiallyrevealed based on various animal tests that energy intake and expressionof IGF, further oncogenesis, have a close relationship. In ratstransplanted with prostate cancer, proliferation of the cancer isinhibited and apoptosis is induced when energy intake is restricted.This effect is correlated with the reduction of IGF-I concentration inblood (Journal of the National Cancer Institute, 91, 512-523, 1999).Similar result has been reported on breast cancer-transplanted mouse,and since the proliferation inhibitory function becomes un-observable bythe administration of IGF-I, it is suggested that IGF-I is taking a mainrole in the proliferation inhibition of cancer by the restriction ofenergy intake (Cancer Research, 57, 4667-4672, 1997).

Relevancy of IGF to cancer has been examined also by clinical andepidemiological studies. It has been reported that IGF-I concentrationin blood plasma and serum is high in breast cancer patients incomparison with healthy persons (European Journal of Cancer, 29A,492-497, 1993, Tumori, 80, 212-215, 1994), and the amount of IGF-IR inbreast cancer tissue is 10 times-higher than that in normal tissue(Cancer Research, 53, 3736-3740, 1993). Also, since the loss ofheterozygosity in IGF-IIR gene was found in about 30% of breast cancerpatients, it was suggested that the IGF-IIR gene has a function as acancer suppressor gene (Breast Cancer Research & Treatment, 47, 269-281,1998). It has been reported that concentrations of IGF-II, IGFBP-2 andIGFBP-3 in sera are high in colon cancer patients in comparison withthose of healthy persons (International Journal of Cancer, 57, 491-497,1994). In addition, it has been shown that serum concentrations ofIGF-II and IGFBP-2 are high in patients of colon adenoma known toprogress to be colon cancer, but these concentrations are reduced by theexcision of adenoma (Journal of Clinical Endocrinology & Metabolism, 85,3402-3408, 2000). Over-expression of IGF-II in gastric cancer tissue hasbeen reported (European Journal of Cancer, 37, 2257-2263, 2001). It hasbeen reported that, in patients of endometrial cancer after menopause,serum IGF-I concentration is high and the IGFBP-1 concentration is lowin comparison with those of healthy persons. On the other hand, adifference was not found regarding the IGFBP-3 concentration (EndocrineJournal, 44, 419-424, 1997). It has been reported that, in patients ofprostate cancer, IGF-I and IGFBP-2 concentrations are high and IGFBP-3concentration is low in sera (British Journal of Cancer, 76, 1115-1118,1997, Urology, 54, 603-606, 1999, Journal of Clinical Endocrinology &Metabolism, 76, 1031-1035, 1993, Journal of Clinical Endocrinology &Metabolism, 77, 299-233, 1993), and production of IGF-II, IGFBP-2,IGFBP-4 and IGFBP-5 is accelerated and production of IGFBP-3 isinhibited in the cancer tissue (Journal of Clinical Endocrinology &Metabolism, 81, 3774-3782, 1996, Journal of Clinical Endocrinology &Metabolism, 81, 411-420, 1996, Journal of Clinical Endocrinology &Metabolism, 81, 3783-3792, 1996). Similar changes in the expression ofIGF-I and IGFBP have been observed also in sera and cancer tissues ofovarian cancer patients (Journal of Clinical Endocrinology & Metabolism,78, 271-276, 1994, Journal of Clinical Endocrinology & Metabolism, 82,2308-2313, 1997, British Journal of Cancer, 73, 1069-1073, 1996).

It has been revealed based on several epidemiological studies that thereis a relevancy between the IGF and IGFBP, and the morbidity risk ofcancer. It has been reported that highness of morbidity risk andhighness of IGF-I concentration in blood and lowness of IGFBP-3concentration in blood show a positive correlation in solid cancers suchas breast cancer, colon cancer, rectum cancer, prostate cancer and lungcancer, that highness of morbidity risk and lowness of IGFBP-3concentration show a positive correlation in infantile leukemia, andthat highness of morbidity risk and highness of the concentration ratioof IGF-I and IGFBP-3 (IGF-I/IGFBP-3) show a positive correlation inbreast cancer (Lancet, 351, 1393-1396, 1998, Science, 279, 563-566,1998, Journal of the National Cancer Institute, 91, 620-625, 1999,Journal of the National Cancer Institute, 91, 151-156, 1999,International Journal of Cancer, 62, 266-270, 1995, Epidemiology 9,570-573, 1998, Breast Cancer Research & Treatment, 47, 111-120, 1998,International Journal of Cancer, 83, 15-17, 1999, International Journalof Cancer, 80, 494-496, 1999, British Journal of Cancer, 76, 1115-1118,1997).

There are reports also on the relevancy of IGF to prognosis of cancer.In the case of breast cancer, it has been reported that expression ofIGF-IR is increased in an estrogen receptor- or progesteronereceptor-positive tissue (Cancer Research, 52, 1036-1039, 1992). Also,there are cases reporting that the prognosis is getting poor by theexpression of IGF-IR (Cancer Research, 57, 3079-3083, 1997, Cancer, 58,1159-1164, 1998). It has also been reported that expression of estrogenreceptor and expression of IGFBP-3 in the tissue have an inversecorrelation (Cancer Research, 52, 5100-5103, 1992, Journal of CellularBiochemistry, 52, 196-205, 1993).

Also, abnormal promotion of IGF function has been found in diabeticcomplications such as diabetic retinopathy and diabetic nephropathy(Science, 276, 1706-1709, 1997, American Journal of Physiology, 274,F1045-F1053, 1998).

In addition, it has been reported that local expression of IGF-I isobserved in rheumatic synovial membrane and also that IGF-I is concernedin the formation of morbid state of reumatoid arthritis (Arthritis &Rheumatism, 32, 66-71, 1989, Journal of Rheumatology, 22, 275-281, 1995,Journal of Clinical Endocrinology & Metabolism, 81, 150-155, 1996,Arthritis & Rheumatism, 39, 1556-1565, 1996).

As described above, the IGF family proteins (IGF, IGF-R, IGFBP)including IGF-I and IGF-II are taking important roles in the oncogenesisand proliferation of cancer and also in diabetic complications andrheumatic arthritis. These facts suggest a possibility of effectingdiagnosis, prevention and treatment of cancers, diabetic complications,rheumtoid arthritis and the like using IGF family proteins as thetarget.

Actually, antitumor effects by inhibiting IGF functions have beenreported (Biochimica et Biophysica Acta, 1332, F105-F126, 1997), forexample that tumorigenicity and metastacity of high metastatic humanbreast cancer cells in mice are reduced and prolongation of survivalperiod is recognized by expressing antisense RNA for IGF-IR (Cancer GeneTherapy, 7, 384-395, 2000), and a report that proliferation of humanrhabdomyosarcoma cell and human breast cancer cell transplanted intomice is inhibited by an anti-IGF-IR antibody (Cancer Research, 54,5531-5534, 1994, Journal of Clinical Investigation, 84, 1418-1423, 1989,Breast Cancer Research & Treatment, 22, 101-106, 1992). On the otherhand, it has been shown that the anti-IGF-IR antibody inhibitsengraftment of a human breast cancer cell showing estrogen-independentgrowth transplanted into mice, but dose not inhibit engraftment of ahuman breast cancer cell showing estrogen-dependent growth orproliferation of the engrafted human breast cancer cell, indicating thatsufficient antitumor effect cannot be obtained by the inhibition ofIGF-IR function alone (Breast Cancer Research & Treatment, 22, 101-106,1992).

Several antibodies are already known as the antibody to IGF (hereinafterreferred to as anti-hIGF antibody). As a typical antibody to human IGF-I(hereinafter referred to as anti-hIGF-I antibody), sm1.2 has beenreported (Proceedings of the National Academy of Sciences of the UnitedStates of America, Vol. 81 (1984) 2389-92. It has been revealed thatsm1.2 has about 5% cross reactivity with hIGF-II, can detect 100 ng ofhIGF-I by western blotting at a concentration of 1 to 2 μg/ml, andinhibits proliferation of a mouse fibroblast cell line BALB/c3T3 by 20ng/ml of hIGF-I at a concentration of 10 to 30 μg/ml (Proceedings of theNational Academy of Sciences of the United Slates of America, Vol. 81(1984) 2389-92, Journal of Clinical Investigation, Vol. 99 (1997) 2961-70.

Val⁵⁹-SmC121 is another anti-hIGF-I antibody, and it has been reportedthat said antibody does not react with human insulin and hIGF-II,recognizes a peptide containing 10th to 12th position Leu-Val-Asp ofhIGF-I, and shows 1 ng/ml of hIGF-I detection sensitivity by aradioimmunoassay using ¹²⁵I-hIGF-I (Journal of endocrinology, 125,327-335, 1990).

It has been reported that an anti-hIGF-I antibody 41/81 has 3% crossreactivity with hIGF-II, and shows 1 ng/ml of hIGF-I detectionsensitivity by a radioimmunoassay using ¹²⁵I-hIGF-I (FEBS Letters, 149,109-112, 1982).

It has been reported that an anti-hIGF-I antibody 35117 has about 0.5%cross reactivity with hIGF-II, can detect 1 μg of hIGF-I by westernblotting at a concentration of 1 μg/ml, entirely inhibits proliferationof a mouse fibroblast cell line BALB/c3T3 by hIGF-I at a concentrationof 12 μg/ml or more, inhibits auto-phosphorylation of hIGF-IR by 1 μg/mlof hIGF-I at a concentration of 30 μg/ml, and shows 0.1 nM of hIGF-Idetection sensitivity by a radioimmunoassay using ¹²⁵I-hIGF-I(Hybridoma, 16, 513-518, 1997).

It has been reported that an anti-hIGF-I antibody BPL-M23 shows abinding activity of 10.5×10⁹ M⁻¹ for hIGF-I, on the other hand, showsrespective cross reactivity of 0.8% and 0.0001% with hIGF-II and humaninsulin, shows reactivity with the IGF of goat, pig, sheep, cattle andrabbit but does not react with the IGF of rat and mouse, and inhibitsfat formation for rat adipocyte by hIGF-I (Journal of MolecularEndocrinology, 2, 201-206, 1989).

It has been reported that anti-hIGF-I antibodies 7A1, 1B3, 4C1 and 5A7recognize different epitopes of the C and D domains of hIGF-I, and showrespective cross reactivity of 6.6%, 0.83%, 12% and 1.2% with hIGF-II(Hybridoma, 12, 737-744, 1993).

It has been reported that 3D1/2/1 reacts with the IGF-I of human andguinea pig but does not react with the IGF-I of rabbit, rat and mouse,and shows a cross reactivity of 7% with hIGF-II (Journal of Clinical ofMetabolism, 54, 474-476, 1982).

As a typical antibody to human IGF-II (hereinafter referred to asanti-hIGF-II antibody), an S1F2 has been reported. It has been revealedthat the S1F2 has a cross reactivity of about 10% with hIGF-I, candetect 10 to 100 ng of hIGF-II by western blotting at a concentration of1 μg/ml, and inhibits the DNA synthesis promoting function of humanfibroblast by 100 ng/ml of hIGF-II at a concentration of 100 μg/ml(Diabetes Research and Clinical Practice, 7, S21-S27, 1989,Endocrinology, 124, 870-877, 1989).

It has been reported that anti-hIGF-II antibodies 2H11, 2B11, ID5 andID9 react with hIGF-II but do not react with hIGF-I, and can determine 1ng/ml of hIGF-II by competitive enzyme immunoassay (hereinafter referredto as ELISA) (Japanese published unexamined application No. 252987/93).

In addition, it is known that when an antibody of a non-human animal,for example a mouse antibody, is administered to human, the administeredmouse antibody is recognized as a foreign body, which induces in thehuman body a human antibody to the mouse antibody (human anti-mouseantibody: hereinafter referred to as HAMA). It is known that the HAMAreacting with the administered mouse antibody to induce side effects(Journal of Clinical Oncology, 2, 881-891, 1984; Blood, 65, 1349-1363,1985; Journal of the National Cancer Institute, 80, 932-936, 1988;Proceedings of the National Academy of Sciences of the United States ofAmerica, 82, 1242-1246, 1985), promotes disappearance of theadministered mouse antibody from the body (Journal of Nuclear Medicine,26, 1011-1023, 1985; Blood, 65, 1349-1363, 1985; Journal of the NationalCancer Institute, 80, 937-942, 1988) and reduces therapeutic effect ofthe mouse antibody (Journal of Immunology, 135, 1530-1535, 1985; CancerResearch 46, 6489-6493, 1986).

In order to solve these problems, attempts have been made to convertantibodies of non-human animals into humanized antibodies such as humanchimeric antibodies and human complementarity determining region(hereinafter referred to as CDR)-grafted antibodies by using generecombination techniques. The human chimeric antibody is an antibodywherein variable region (hereinafter referred to as V region) of theantibody is an antibody of a non-human animal and constant region(hereinafter referred to as C region) is a human antibody (Proceedingsof the National Academy of Sciences of the United States of America, 81,6851-6855, 1984), and the human CDR-grafted antibody is an antibodywherein amino acid sequence of CDR in the V region of an antibody of anon-human animal is grafted to an appropriate position of a humanantibody (Nature, 321, 522-525, 1986). In comparison with antibodies ofnon-human animals such as mouse antibody, these humanized antibodies aremore advantageous in clinical applications to human. For example,regarding immunogenicity and stability in blood, it has been reportedthat blood half-life of a human chimeric antibody was extended about 6times in comparison with a mouse antibody when administered to human(Proceeding of the National Academy of Sciences of the United States ofAmerica, 86, 4220-4224, 1989). As to a human CDR-grafted antibody, ithas been reported that its immunogenicity was reduced and bloodhalf-life was extended in comparison with a mouse antibody in a studyusing a monkey (Cancer Research, 56, 1118-1125, 1996; Immunology, 85,668-674, 1995). Thus, it is expected that humanized antibodies have lessside effects in comparison with antibodies of non-human animals, andtheir-therapeutic effects are maintained for a long period of time.Further, humanized antibodies are prepared by using gene recombinationtechniques, and they can be prepared as various forms of molecules. Forexample, when a γ-1 subclass is used as the heavy chain (hereinafterreferred to as H chain) C region of a human antibody, a humanizedantibody which is stable in blood and has high effector activities suchas antibody-dependent cellular cytotoxicity and the like can be prepared(Cancer Research, 56, 1118-1125, 1996). A humanized antibody having higheffector activity is markedly useful when destruction of targets such ascancer is desired. On the other hand, in the case that merely atarget-neutralizing function alone is required, or in the case thatthere is a possibility of causing a side effect due to destruction of atarget by an effector activity, a γ4 subclass is suitably used as the Hchain C region of a human antibody, because γ4 subclass generally haslow effector activity (Journal of Experimental Medicine, 166, 1351-1361,1987; Journal of Experimental Medicine, 168, 127-142, 1988), and sideeffects can be avoided, and further extension of blood half-life incomparison with a mouse antibody can be expected (Immunology, 85,668-674, 1995). In addition, with the recent advances in proteinengineering and genetic engineering, it became possible to prepareantibody fragments having more smaller molecular weight such as Fab,Fab′, F(ab′)₂, scFv (Science, 242, 423-426, 1988), dsFv (MolecularImmunology, 32, 249-258, 1995) and CDR-containing peptide (Journal ofBiological Chemistry, 271, 2966-2971, 1996) from antibodies includinghumanized antibodies. Since these antibody fragments have smallermolecular weight in comparison with whole antibody molecules, they havesuperior transferring property to target tissues (Cancer Research, 52,3402-3408, 1992).

Based on the above, the IGF family proteins which take important rolesin the oncogenesis and proliferation of cancer and also in diabeticcomplications and rheumatoid arthritis are controlling these diseasesthrough complicated entanglement of growth factors including insulin,IGF-I and IGF-II, receptors including insulin receptor, IGF-IR andIGF-IIR and IGFBP. Accordingly, it is difficult to suppress thesediseases completely by inhibiting a part of these interactions. Thoughthere are many reports on antibodies which recognize IGF-I and/or IGF-IIconsidered to be useful as medicament, there are no reports onantibodies which can simultaneously inhibit functions of IGF-I andIGF-II by strongly binding to IGF-I and IGF-II.

In addition, as antibodies to be used for the clinical application tohuman, humanized antibodies are desirable than antibodies of a non-humananimal such as mouse antibody. However, there are no reports on thepreparation of recombinant antibodies such as humanized antibody as ananti-hIGF antibody, and also on antibody fragments thereof.

DISCLOSURE OF THE INVENTION

It is known that hIGF family-mediated cell-growth is working in varioukinds of cancer cells, and it is expected that inhibition ofhIGF-mediated signal transduction, in the case it is attainable, iseffective for treating diseases such as proliferation and metastasis ofsolid cancers, diabetic complications and rheumatoid arthritis in human.

An object of the present invention is to obtain a substance whichinhibits cell growth via IGF by blocking hIGF family-mediated signaltransduction, and to further provide application methods of saidsubstance.

The present invention relates to the following (1) to (24).

(1) An antibody or an antibody fragment thereof, which specificallybinds to IGF-I and IGF-II to inhibit functions of human IGF-I and humanIGF-II and has the binding activity with a binding constant of 5×10⁹ M⁻¹or more measured with a biosensor BIACORE.

(2) The antibody or the antibody fragment thereof according to the above(1), wherein the binding activity to human IGF-I and the bindingactivity to human IGF-II are the same degree.

(3) The antibody or the antibody fragment thereof according to the above(1) or (2), wherein CDR1, CDR2 and CDR3 of heavy chain variable region(VH) of an antibody or an antibody fragment thereof comprise amino acidsequences represented by SEQ ID NOS: 5, 6 and 7 respectively, and/orCDR1, CDR2 and CDR3 of light chain variable region (VL) of the antibodyor an antibody fragment comprise amino acid sequences represented by SEQID NOS: 8, 9 and 10 respectively.

(4) The antibody or the antibody fragment thereof according to any oneof the above (1) to (3), wherein the antibody is an antibody of anon-human animal or a recombinant antibody.

(5) The antibody or the antibody fragment thereof according to the above(4), wherein the recombinant antibody is selected from the groupconsisting of a human chimeric antibody, a human CDR-grafted antibodyand a human antibody.

(6) The antibody or the antibody fragment thereof according to the above(4), wherein VH of the antibody of a non-human animal comprises theamino acid sequence represented by SEQ ID NO: 2, and/or VL of theantibody of a non-human animal somprises the amino acid sequencerepresented by SEQ ID NO: 4.

(7) The antibody or the antibody fragment thereof according to the above(3) or (6), wherein the antibody of a non-human animal is produced by ahybridoma KM1468 (FERM BP-7978).

(8) The antibody or the antibody fragment thereof according to the above(5), wherein VH of the human chimeric antibody comprises the amino acidsequence represented by SEQ ID NO: 2, and/or VL of the human chimericantibody comprises the amino acid sequence represented by SEQ ID NO: 4.

(9) The antibody or the antibody fragment thereof according to the above(5) or (8), wherein the human chimeric antibody comprises VH and/or VLof the antibody produced by KM1468 (FERM BP-7978).

(10) The antibody or the antibody fragment thereof according to any oneof the above (5), (8) and (9), wherein the human chimeric antibodycomprises a constant region of a human antibody.

(11) The antibody or the antibody fragment thereof according to theabove (10), wherein the constant region of a human antibody comprisesthe constant region of a human antibody IgG1 class and/or κ class.

(12) The antibody or the antibody fragment thereof according to any oneof the above (5) and (8) to (11), wherein the human chimeric antibody isproduced by a transformant KM3002 (FERM BP-7996).

(13) The antibody or the antibody fragment thereof according to theabove (4), wherein CDR1, CDR2 and CDR3 of VH of the human CDR-graftedantibody comprises the amino acid sequences represented by SEQ ID NOS:5, 6 and 7 respectively, and/or CDR1, CDR2 and CDR3 of VL of the humanCDR-grafted antibody comprises the amino acid sequences of SEQ ID NOS:8, 9 and 10 respectively.

(14) The antibody or the antibody fragment thereof according to theabove (5) or (13), wherein the human CDR-grafted antibody comprises CDRof VH of the antibody produced by KM1468 (FERM BP-7978) and/or CDR of VLof the antibody produced by KM1468 (FERM BP-7978).

(15) The antibody or the antibody fragment thereof according to any oneof the above (4), (13) and (14), wherein the human CDR-grafted antibodycomprises a constant region of a human antibody.

(16) The antibody or the antibody fragment thereof according to theabove (15), wherein the constant region of a human antibody comprisesthe constant region of a human antibody IgG1 class and/or κ class.

(17) The antibody fragment according to any one of the above (1) to(16), wherein the antibody fragment is selected from the groupconsisting of Fab, Fab′, F(ab′)₂, single chain antibody (scFv),dimerized V region (diabody), disulfide-stabilized V region (dsFv) and aCDR-containing peptide.

(18) A DNA coding for the antibody or antibody fragment thereofaccording to any one of the above (1) to (17).

(19) A recombinant vector which contains the DNA according to the above(18).

(20) A transformant which is obtained by introducing the recombinantvector according to the above (19) into a host cell.

(21) A method for producing an antibody or the antibody fragmentthereof, which comprises culturing the transformant according to theabove (20) in a medium to produce and accumulate the antibody or theantibody fragment thereof according to any one of the above (1) to (16)in a culture, and recovering the antibody or the antibody fragmentthereof from the culture.

(22) A medicament which comprises at least one of the antibody and theantibody fragment thereof according to any one of the above (1) to (17)as the active ingredient.

(23) A therapeutic agent against a human IGF-related disease or adisease whose morbid state progresses by abnormal promotion of human IGFproduction, which comprises at least one of the antibody and theantibody fragment thereof according to any one of the above (1) to (17)as the active ingredient.

(24) A diagnostic agent for a human IGF-related disease or a diseasewhose morbid state progresses by abnormal promotion of human IGFproduction, which comprises at least one of the antibody and an antibodyfragment thereof according to any one of the above (1) to (17).

Examples of the anti-hIGF antibody of the present invention include anantibody specifically binds to human IGF-I and human IGF-II to inhibitfunctions of human IGF-I and human IGF-II which has the binding activitywith a binding constant of 5×10⁹ M⁻¹ or more measured with a biosensorBIACORE, and particularly desirable is an antibody in which the bindingactivity to hIGF-I and the binding activity to hIGF-II are almost thesame and which inhibits functions of hIGF-I and hIGF-II.

The term “the binding activity to hIGF-I and the binding activity tohIGF-II are almost the same” means that the antibody can bind to bothhIGF-I and hIGF-II equivalently. The equivalent binding can berepresented as the relative value by numerating binding activity of theantibody to hIGF-I or hIGF-II. The equivalent binding activity meansthat when the binding activity of the antibody to hIGF-I is defined as1, the binding activity to hIGF-II is 0.1 to 10, preferably 0.2 to 5,more preferably 0.5 to 2, most preferably 1. Examples of the index ofthe binding activity include a binding constant (hereinafter referredalso to as K_(A)) measured by a biosensor method which uses theprinciple of surface plasmon resonance or the like (hereinafter referredto as biosensor BIACORE).

Examples of the antibody of the present invention include an antibodywhich recognizes an epitope existing in natural type hIGF-I and hIGF-IIand an antibody which recognizes the three-dimensional structure ofnatural type hIGF-I and hIGF-II. The examples further include anantibody which show cross reactivity with IGF of non-human organism.

Regarding the function of hIGF-I and hIGF-II, it may be any function inwhich hIGF-I and hIGF-II are concerned, such as control ofproliferation, differentiation or apoptosis of epithelial cells of thebreast, prostate, lungs, colon and the like.

Examples of the anti-hIGF antibody of the present invention include anantibody of a non-human animal, a recombinant antibody and an antibodyfragment thereof.

Examples of the antibody of a non-human animal include a polyclonalantibody and a monoclonal antibody, preferable is a monoclonal antibody.

The monoclonal antibody of a non-human animal according to the presentinvention can be obtained by immunizing a non-human animal with hIGF,preparing hybridomas from an antibody-producing cell of the immunizedanimal and a myeloma cell, selecting a monoclonal hybridoma, culturingthe monoclonal hybridoma and then purifying it from the culturesupernatant. As the non-human animal, any one of mouse, rat, hamster,rabbit and the like can be used with the proviso that hybridomas can beprepared therefrom.

Preferred examples of the antibody of the present invention includeantibodies in which CDR1, CDR2 and CDR3 of VH comprise amino acidsequences of SEQ ID NOS: 5, 6 and 7 respectively, and/or CDR1, CDR2 andCDR3 of VL comprise the sequences of SEQ ID NOS: 8, 9 and 10respectively.

Specific examples of the monoclonal antibody of a non-human animalaccording to the present invention include a rat antibody KM1468 whichis produced by a hybridoma KM1468 (FERM BP-7978).

Examples of the recombinant antibody of the present invention include ahumanized antibody, a human antibody and the like.

Examples of the humanized antibody include a human chimeric antibody, ahuman CDR-grafted antibody and the like.

The human chimeric antibody is an antibody which comprises VH and VL ofan antibody of a non-human animal and CH and CL of a human antibody.

The human chimeric antibody of the present invention can be produced bypreparing cDNAs coding for VH and VL from a hybridoma capable ofproducing a monoclonal antibody which has the ability to inhibitfunctions of human IGF-I and human IGF-II by specifically binding tohuman IGF-I and human IGF-II and has the binding activity with a bindingconstant of 5×10⁹ M⁻¹ or more measured with a biosensor BIACORE,constructing a human chimeric antibody expression vector by respectivelyinserting the cDNAs into an expression vector for animal cell use havinggenes coding for CH and CL of a human antibody, and then expressing byintroducing the vector into an animal cell.

The CH of the human chimeric antibody may be any region with the provisothat it belongs to human immunoglobulin (hereinafter referred to ashIg), but preferably an hIgG class, and anyone of hIgG1, hIgG2, hIgG3and hIgG4 subclasses belonging to the hIgG class can be used. Also, theCL of the human chimeric antibody may be any region with the provisothat it belongs to the hIg, and those of κ class or λ class can be used.

Examples of the human chimeric antibody which binds to hIGF-I andhIGF-II according to the present invention (to be referred to asanti-hIGF chimeric antibody hereinafter) include an anti-hIGF chimericantibody containing antibody VH CDR1, CDR2 and CDR3, respectivelycomprising the amino acid sequences represented by SEQ ID NOS: 5, 6 and7 and/or VL CDR1, CDR2 and CDR3, respectively comprising the amino acidsequences represented by SEQ ID NOS: 8, 9 and 10, an anti-hIGF chimericantibody containing VH and/or VL of the monoclonal antibody produced bythe hybridoma KM1468, an anti-hIGF chimeric antibody in which theantibody VH contains the 1st to 118th positions of the amino acidsequence represented by SEQ ID NO: 2 and/or the VL contains the 1st to107th positions of the amino acid sequence represented by SEQ ID NO: 4,and an anti-hIGF chimeric antibody in which the antibody VH comprisesthe amino acid sequence represented by SEQ ID NO: 2, the human antibodyCH comprises an amino acid sequence of hIgG1 subclass, the antibody VLcomprises the amino acid sequence represented by SEQ ID NO: 4 and thehuman antibody CL comprises an amino acid sequence of κ class, andspecifically, the anti-hIGF chimeric antibody KM3002 produced by atransformant KM3002 (FERM BP-7996) is mentioned. Antibodies in which oneor more amino acids of these amino acid sequences are deleted, added,substituted or inserted, and which have the ability to inhibit functionsof human IGF-I and human IGF-II by specifically binding to human IGF-Iand human IGF-II and have the binding activity with a binding constantof 5×10⁹ M⁻¹ or more measured with a biosensor BIACORE are also includedin the antibody of the invention.

The human CDR-grafted antibody is an antibody in which CDR amino acidsequences of VH and VL of an antibody of a non-human animal are graftedto appropriate positions of VH and VL of a human antibody.

The human CDR-grafted antibody of the present invention can be producedby constructing cDNAs coding for V regions prepared by grafting CDRamino acid sequences of VH and VL of an antibody of a non-human animal,which has the ability to inhibit functions of human IGF-I and humanIGF-II by specifically binding to human IGF-I and human IGF-II and hasthe binding activity with a binding constant of 5×10⁹ M⁻¹ or moremeasured with a biosensor BIACORE, to the FR of VH and VL of an optionalhuman antibody, constructing a human CDR-grafted antibody expressionvector by respectively inserting the cDNAs into an expression vector foranimal cell use having a gene coding for CH and CL of a human antibody,and then expressing by introducing the vector into an animal cell.

The CH of the human CDR-grafted antibody may be any region with theproviso that it belongs to hIg, but preferably an hIgG class, and anyone of hIgG1, hIgG2, hIgG3 and hIgG4 subclasses belonging to the hIgGclass can be used. Also, the CL of the human CDR-grafted antibody may beany region with the proviso that it belongs to the hIg, and those of κclass or λ class can be used.

Examples of the human CDR-grafted antibody which binds to hIGF-I andhIGF-II according to the present invention (hereinafter referred to asanti-hIGF CDR-grafted antibody) include an anti-hIGF CDR-graftedantibody containing antibody VH CDR1, CDR2 and CDR3, respectivelycomprising the amino acid sequences represented by SEQ ID NOS: 5, 6 and7 and/or VL CDR1, CDR2 and CDR3, respectively comprising the amino acidsequences represented by SEQ ID NOS: 8, 9 and 10, an anti-hIGFCDR-grafted antibody containing VH CDR and/or VL CDR of the monoclonalantibody produced by the hybridoma KM1468, an anti-hIGF CDR-graftedantibody in which the antibody VH contains the 1st to 118th positions ofthe amino acid sequence represented by SEQ ID NO: 15 and/or the VLcontains the 1st to 107th positions of the amino acid sequencerepresented by SEQ ID NO: 16, and an anti-hIGF CDR-grafted antibody inwhich the antibody VH comprises the amino acid sequence represented bySEQ ID NO: 15, the human antibody CH comprises an amino acid sequence ofhIgG1 subclass, the antibody VL comprises the amino acid sequencerepresented by SEQ ID NO: 16 and the human antibody CL comprises anamino acid sequence of κ class. Antibodies in which one or more aminoacids of these amino acid sequences are deleted, added, substituted orinserted, and which have the ability to inhibit functions of human IGF-Iand human IGF-II by specifically binding to human IGF-I and human IGF-IIand have the binding activity with a binding constant of 5×10⁹ M⁻¹ ormore measured with a biosensor BIACORE are also included in the antibodyof the present invention.

Though the human antibody generally means an antibody naturally existedin the human body, it also includes antibodies obtained from a humanantibody phage library and a human antibody-producing transgenic animalprepared based on the recent advance in the genetic engineering, cellengineering and embryo engineering techniques.

Regarding the antibody existed in the human body, for example, alymphocyte capable of producing said antibody can be cultured byisolating a human peripheral lymphocyte, immortalizing by infecting withEB virus or the like and then cloning, and said antibody can be purifiedfrom the culture supernatant.

The human antibody phage library is a library in which antibodyfragments of Fab, scFv and the like are expressed on the phage surfaceby inserting an antibody gene prepared from human B cell into a phagegene. A phage expressing antibody, fragments having desired antigenbinding activity on the surface can be recovered from said library usingthe binding activity to an antigen-immobilized substrate as an index.Said antibody fragments can be further converted into a human antibodymolecule comprising two full length H chains and two full length Lchains by genetic engineering techniques.

The human antibody-producing transgenic animal means an animal in whicha human antibody gene is integrated into its cells. For example, a humanantibody-producing transgenic mouse can be prepared by introducing ahuman antibody gene into a mouse ES cell, transplanting said ES cellinto early embryo of a mouse and then developing. Regarding the methodfor preparing a human antibody from a human antibody-producingtransgenic animal, the human antibody can be produced and accumulated ina culture supernatant by culturing a human antibody-producing hybridomaobtained by a hybridoma preparation method generally carried out innon-human animals.

As the antibody fragments of the present invention, Fab, Fab′, F(ab′)₂,scFv, diabody, dsFv, a CDR-containing peptide and the like can beexemplified.

Among fragments obtained by treating IgG with a protease papain(digested at the 224th amino acid residue of H chain), Fab is anantibody fragment of about 50,000 in molecular weight having an antigenbinding activity in which about half of the H chain N-terminal side andfull length L chain are bonded through disulfide bond.

The Fab of the present invention can be prepared by treating an antibodywhich binds to hIGF-I and hIGF-II with a protease papain. Alternatively,Fab can be produced by inserting a DNA coding for the Fab of saidantibody into an expression vector for prokaryote or an expressionvector for eucaryote and expressing by introducing said vector into aprokaryote or a eucaryote.

Among fragments obtained by treating IgG with a protease pepsin(digested at the 234th amino acid residue of H chain), F(ab′)₂ is anantibody fragment of about 100,000 in molecular weight having an antigenbinding activity, which is slightly larger than a product in which Fabfragments are bonded via disulfide bond of the hinge region.

The F(ab′)₂ of the present invention can be prepared by treating anantibody which binds to hIGF-I and hIGF-II with a protease pepsin.Alternatively, it can be prepared by carrying out thioether bonding ordisulfide bonding of the Fab′ fragments described below.

Fab′ is an antibody fragment of about 50,000 in molecular weight havingan antigen binding activity obtained by digesting the hinge regiondisulfide bond of the aforementioned F(ab′)₂.

The Fab′ of the present invention can be obtained by treating theF(ab′)₂ of the present invention which binds to hIGF-I and hIGF-II witha reducing agent dithiothreitol. Alternatively, Fab′ can be produced byinserting a DNA coding for the Fab′ fragment of said antibody into anexpression vector for prokaryote or an expression vector for eucaryoteand expressing by introducing said vector into a prokaryote or aeucaryote.

The scFv is an antibody fragment having an antigen binding activity,which is a VH-P-VL or VL-P-VH polypeptide prepared by connecting one VHand one VL using an appropriate peptide linker (to be referred to as Phereinafter).

The scFv of the present invention can be obtained by preparing cDNAscoding for VH and VL of the antibody of the present invention whichbinds to hIGF-I and hIGF-II, thereby constructing a DNA coding for scFv,inserting said DNA into an expression vector for prokaryote or anexpression vector for eucaryote and expressing by introducing saidexpression vector into a prokaryote or a eucaryote.

Diabody is an antibody fragment in which scFv is dimerized and which hasdivalent antigen binding activities. The divalent antigen bindingactivities may be the same, or one of them can be used as a differentantigen binding activity.

The diabody of the present invention can be obtained by preparing cDNAscoding for VH and VL of the antibody of the present invention whichbinds to hIGF-I and hIGF-II, constructing a DNA coding for scFv in sucha manner that length of the amino acid sequence of P becomes 8 residuesor less, inserting said DNA into an expression vector for prokaryote oran expression vector for eucaryote and expressing by introducing saidexpression vector into a prokaryote or a eucaryote.

The dsFv is a product in which polypeptides prepared by replacing oneamino acid residue in VH and one in VL by cysteine residues are bondedvia the disulfide bond between said cysteine residues. The amino acidresidues to be replaced by cysteine residues can be selected based onthe estimation of three-dimensional structure of the antibody inaccordance with the method shown by Reiter et al. (Protein Engineering,7, 697-704, 1994).

The dsFv of the present invention can be obtained by preparing cDNAscoding for VH and VL of the antibody of the present invention whichbinds to hIGF-I hIGF-II, constructing a DNA coding for dsFv, insertingsaid DNA into an expression vector for prokaryote or an expressionvector for eucaryote and expressing by introducing said expressionvector into a prokaryote or a eucaryote.

A CDR-containing peptide comprises at least one or more of CDR of VH orVL. Peptides containing plural of CDRs can be linked directly or via anappropriate peptide linker.

The CDR-containing peptide of the present invention can be obtained byconstructing a DNA coding for CDRs of VH and VL of the antibody of theinvention which binds to hIGF-I and hIGF-II, inserting said DNA into anexpression vector for prokaryote or an expression vector for eucaryoteand expressing by introducing said expression vector into a prokaryoteor a eucaryote.

The CDR-containing peptide can also be produced by chemical synthesismethod such as Fmoc method (fluorenylmethoxycarbonyl method) and tBocmethod (t-butyloxycarbonyl method.

Antibody derivatives prepared by binding a radioisotope, a low moleculardrug, a high molecular drug, a protein and the like, chemically or by agenetic engineering technique, to the antibody of the invention whichbinds to hIGF-I and hIGF-II and antibody fragments thereof are includedin the antibody of the present invention.

Derivatives of the antibody of the present invention can be produced bybinding a radioisotope, a low molecular drug, a high molecular drug, aprotein and the like to H chain or L chain N-terminal side or C-terminalside of the antibody of the present invention which binds to hIGF-I andhIGF-II and antibody fragments thereof, an appropriate substituent groupor side chain of the antibody and antibody fragments or a sugar chain inthe antibody and antibody fragments, by chemical techniques(Introduction to Antibody Engineering, written by O. Kanemitsu,published by Chijin Shokan, 1994).

They can also be produced by connecting a DNA coding for the antibody ofthe present invention which binds to hIGF-I and hIGF-II or an antibodyfragment thereof and a DNA coding for a protein to be bonded andinserting into an expression vector, and expressing by introducing saidexpression vector into an appropriate host cell.

As the radioisotope, ¹³¹I, ¹²⁵I and the like can be exemplified, andthey can be connected to the antibody for example by the chloramine Tmethod.

Examples of the low molecular drug include antitumor agents such asalkylating agents including nitrogen mustard, cyclophosphamide and thelike, metabolic antagonists including 5-fluorouracil, methotrexate andthe like, antibiotics including daunomycin, bleomycin, mitomycin C,daunorubicin, doxorubicin and the like, plant alkaloids includingvincristine, vinblastine, vindesine and the like, and hormone agentsincluding tamoxifen, dexamethasone and the like (Clinical Tumor Science,edited by Japan Clinical Tumor Research Association, published by Gan toKagaku Ryoho-sha, 1996), or anti-inflammatory agents such as steroidagents including hydrocortisone, prednisone and the like, non-steroidalagents including aspirin, indometacin and the like, immunomodulatorsincluding aurothiomalate, penicillamine and the like, immunosuppressantsincluding cyclophosphamide, azathioprine and the like, andanti-inflammatory such as antihistaminics including chlorpheniraminemaleate, clemastine and the like (Inflammation and Anti-inflammationTherapy, published by Ishiyaku Shuppan, 1982). For example, as a methodfor connecting daunomycin and an antibody, a method in which aminogroups of daunomycin and the antibody are connected via glutaraldehydeand a method in which amino group of daunomycin and carboxyl group ofthe antibody are connected via a water-soluble carbodiimide can beexemplified.

Examples of the high molecular drug include polyethylene glycol (to bereferred to as PEG hereinafter), albumin, dextran, polyoxyethylene,styrene maleic acid copolymer, polyvinyl pyrrolidone, pyran copolymer,hydroxypropylmethacrylamide and the like. By connecting these highmolecular compounds to antibodies and antibody fragments, variouseffects can be expected, for example, (1) improvement of stability forvarious chemical, physical or biological factors, (2) significantextension of blood half-life, (3) disappearance of immunogenicity andsuppression of antibody production (Bioconjugate Medicaments, publishedby Hirokawa Shoten, 1993). For example, as a method for connecting PEGto an antibody, a method in which they are allowed to undergo thereaction with a PEG modifying agent can be exemplified (BioconjugateMedicaments, published by Hirokawa Shoten, 1993). Examples of the PEGmodifying agent include an agent for modifying ε-amino group of lysine(Japanese published unexamined application No. 178926/86), an agent formodifying carboxyl group of aspartic acid and glutamic acid (Japanesepublished unexamined application No. 23587/81), an agent for modifyingguanidino group of arginine (Japanese published unexamined applicationNo. 117920/90) and the like.

Examples of the protein include cytokines which activate immunocompetentcells such as human interleukin 2 (hereinafter referred to as hIL-2),human granulocyte macrophage colony-stimulating factor (hereinafterreferred to as hGM-CSF), human macrophage colony-stimulating factor (tobe referred to as hM-CSF hereinafter), human interleukin 12 (hereinafterreferred to as hIL-12) and the like. In addition, toxins having theactivity to directly damage cancer cells such as ricin and diphtheriatoxin can also be used. For example, in the case of a fusion antibodywith a protein, the fusion antibody can be produced by connecting a cDNAcoding for a protein to another cDNA coding for an antibody or antibodyfragment, thereby constructing a DNA coding for a fusion antibody,inserting said DNA into an expression vector for prokaryote or anexpression vector for eucaryote and expressing by introducing saidexpression vector into a prokaryote or a eucaryote.

Regarding the antibody of the present invention to hIGF-I and hIGF-IIand antibody fragments thereof, their binding activity to hIGF-I andhIGF-II and activity to inhibit functions of hIGF-I and hIGF-II can beevaluated by measuring ELISA (Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Chapter 14, 1988; Monoclonal Antibodies:Principles and Practice, Academic Press Limited, 1996), K_(A) measuredwith a biosensor BIACORE (Journal of Immunological Methods, 145,229-240, 1991), inhibitory activity for cell proliferation by hIGF-I andhIGF-II (Cancer Research, 48, 4083-4092, 1988) and the like.

As the diseases in which the hIGF of the present invention is concernedand diseases in which their morbid states progress by abnormal promotionof hIGF production, any disease is included with the proviso that it isa disease in which its morbid state progresses by abnormal cellproliferation caused by hIGF, regardless of mild or serious-illness, andthe specific examples include cancer, diabetic complication, rheumatoidarthritis and the like.

The following describes preparation method and activity evaluation of anantibody or an antibody fragment thereof which specifically binds tohuman IGF-I and human IGF-II, has the ability to inhibit functions ofhuman IGF-I and human IGF-II and has binding activity with a bindingconstant of 5×10⁹ M⁻¹ or more measured with a biosensor BIACORE.

1. Preparation of Anti-hIGF Monoclonal Antibody of a Non-Human Animal

(1) Preparation of Antigen

A recombinant hIGF protein is obtained by introducing an expressionvector containing a cDNA coding for hIGF into Escherichia coli, a yeast,an insect cell, an animal cell or the like and expressing the proteintherein. Alternatively, a synthetic peptide having an hIGF partialsequence can also be used as the antigen.

As the partial peptide for antigen, a protein partial sequence ofapproximately 5 to 30 residues is selected. For obtaining an antibodywhich recognizes said protein under a state of having non-denaturednatural structure, it is necessary to select, as an antigen peptide, apartial sequence existing on the protein surface in view ofthree-dimensional structure. The moiety existing on the protein surfacein view of three-dimensional structure can be assumed by estimating apartial sequence being high hydrophilic using a commercially availableprotein sequence analyzing software such as Genetyx Mac. That is, thisis because generally a low hydrophilic region is present in inner partof protein in view of three-dimensional structure in many cases, while ahigh hydrophilic region is present on the protein surface. In addition,N-terminus and C-terminus of a protein are present on the proteinsurface in many cases. However, the partial peptide selected in thismanner is not always used as an antigen which establishes desiredantibody.

Cysteine is added to a terminus of the partial peptide to crosslink witha protein. When an inner sequence of the protein is selected, N-terminusof the peptide is acetylated and the C-terminus is subjected toamidation as required.

The partial peptide can be synthesized by a general liquid phase andsolid phase peptide synthesis methods, a method in which these areoptionally combined or a method in accordance therewith (The Peptides,Analysis, Synthesis, Biology, Vol. 1, 1979; Vol. 2, 1980; Vol. 3, 1981,Academic Press; Basics and Experimentations of Peptide Synthesis,Maruzen, 1985; Development of Medicaments, a second series, Vol. 14,Peptide Synthesis, Hirokawa Shoten, 1991; International Journal ofPeptide & Protein Research, 35, 161-214, 1990).

In addition, an automatic peptide synthesizer can also be used.Synthesis of a peptide by a peptide synthesizer can be carried out on acommercially available peptide synthesizer such as a peptide synthesizermanufactured by Shimadzu, a peptide synthesizer manufactured by AppliedBiosystems, Inc. (hereinafter referred to as ABI), a peptide synthesizermanufactured by Advanced ChemTech Inc. (hereinafter referred to as ACT)or the like, in accordance with each synthesis program usingNα-Fmoc-amino acids, Nα-Boc-amino acids or the like whose side chainsare properly protected.

The protected amino acids to be used as the material and carrier resinscan be purchased from ABI, Shimadzu, Kokusan Kagaku, Nova Biochem,Watanabe Kagaku, ACT, Peptide Research Laboratory, etc. In addition, theprotected amino acids to be used as the material, protected organicacids and protected organic amines can be synthesized by reportedsynthesizing methods or in accordance therewith (The Peptides, Analysis,Synthesis, Biology, Vol. 1, 1979; Vol. 2, 1980; Vol. 3, 1981, AcademicPress; Basics and Experimentations of Peptide Synthesis, Maruzen, 1985;Development of Medicaments, a second series, Vol. 14, Peptide Synthesis,Hirokawa Shoten, 1991; International Journal of Peptide & ProteinResearch, 35, 161-214, 1990).

(2) Immunization of Animal and Preparation of Antibody Producing Cells

Any one of mouse, rat, hamster, rabbit ant the like can be used as theanimal to be used in the immunization, with the proviso that a hybridomacan be prepared therefrom. An example which uses mouse and rat isdescribed below.

Mice or rats of 3 to 20 weeks of age are immunized with the antigenprepared in the above 1(1), and antibody producing cells are collectedfrom the spleen, lymph node or peripheral blood of the animals. Theimmunization is carried out several times by administering the antigentogether with an appropriate adjuvant to the animals subcutaneously,intravenously or intraperitoneally. Examples of the adjuvant includeFreund's complete adjuvant and aluminum hydroxide gel plus pertussisvaccine can be cited. Also, a conjugate with carrier protein such asbovine serum albumin (hereinafter referred to as BSA) and keyhole limpethemocyanin (hereinafter referred to as KLH) can be prepared and used asthe immunogen. Three to seven days after each administration of antigen,a blood sample is taken from the venous plexus of the fundus of the eyeor from the tail vein of each immunized animal, the sample is tested asto whether it is reactive with the hIGF used as the antigen by ELISA orthe like, and a mouse or rat whose serum shows a sufficient antibodytiter is used as a supply source of antibody producing cells. On the 3rdto 7th day after final administration of the antigen, the spleen or thelike is excised from the immunized mouse or rat in accordance with aknown method (Antibodies—A Laboratory Manual, Cold Spring HarborLaboratory, 1988), and the antibody producing cells are fused withmyeloma cells.

(3) Preparation of Myeloma Cells

As the myeloma cells, any myeloma cell which can proliferate in vitrocan be used, such as the 8-azaguanine-resistant mouse derived myelomacell lines P3-X63Ag8-U1 (P3-U1) (European Journal of Immunology, 6,511-519, 1976), SP2/O-Ag14 (SP-2) (Nature, 276, 269-270, 1978),P3-X63-Ag8653 (653) (Journal of Immunology, 123, 1548-1550, 1979) orP3-X63-Ag8 (X63) (Nature, 256, 495-497, 1975). Regarding culturing andsub-culturing of these cell strains, 2×10⁷ or more of the cells areprepared until the time of cell fusion in accordance with a known method(Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

(4) Cell Fusion

The antibody producing cells and myeloma cells obtained in the above arewashed, mixed with cell aggregating medium such as polyethyleneglycol-1000 (to be referred to as PEG-1000 hereinafter) to effect fusionof the cells and then suspended in a medium. Modified Eagle's medium(hereinafter referred to as MEM), phosphate buffered saline (hereinafterreferred to as PBS) or the like is used for the washing of cells. Also,in order to selectively obtain a fused cell of interest, HAT medium{normal medium [a medium prepared by adding 1.5 mM glutamine, 50 μM2-mercaptoethanol, 10 μg/ml gentamicin and 10% fetal calf serum(hereinafter referred to as FCS) to RPMI-1640 medium] furthersupplemented with 0.1 mM hypoxanthine, 15 μM thymidine and 0.4 μMaminopterin} is used as the medium in which fused cells are to besuspended.

After the culturing, a portion of the culture supernatant is taken, anda sample which reacts with the antigen protein but does not react withnon-antigen proteins is selected by ELISA. Next, single cell cloning iscarried out by limiting dilution method, and a cell showing stably highantibody titer by ELISA is selected as a monoclonal antibody producinghybridoma.

(5) Selection of Hybridoma

Selection of a hybridoma which produces an anti-hIGF monoclonal antibodyis carried out in accordance with a known method (Antibodies—ALaboratory Manual, Cold Spring Harbor Laboratory, 1988) by the ELISAdescribed in the following. These methods render possible measurement ofthe binding activity of antibodies contained in the culture supernatantsof transformants which produce the anti-hIGF chimeric antibody,anti-hIGF CDR-grafted antibody or an antibody fragment thereof whichwill be described later, or of all of purified antibodies.

ELISA

An antigen is immobilized on a 96 well ELISA plate and allowed to reactwith a culture supernatant of a hybridoma or a purified antibody as aprimary antibody.

After the reaction with the primary antibody, the plate is washed and asecondary antibody is added thereto. As the secondary antibody, anantibody capable of recognizing the primary antibody and labeled withbiotin, an enzyme, a chemiluminescence substance, a radioisotope or thelike is used. Specifically, an antibody capable of recognizing a mouseantibody is used as the secondary antibody when mouse was used inpreparing hybridoma, or an antibody capable of recognizing a ratantibody is used as the secondary antibody when rat was used inpreparing hybridoma.

After the reaction, a reaction is carried out in response to thelabeling agent of the secondary antibody, and a sample whichspecifically reacts with the antigen is selected as a monoclonalantibody producing hybridoma.

Hybridoma KM1468 and the like can be cited as specific examples of saidhybridoma. The hybridoma KM1468 was deposited on Mar. 26, 2002, as FERMBP-7978 in International Patent Organism Depositary, National Instituteof Advanced Industrial Science and Technology (postal code 305-8566;Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan).

(6) Purification of Monoclonal Antibody

The anti-hIGF monoclonal antibody producing hybridoma cells obtained in1(4) are intraperitoneally injected into 8 to 10-week-old mice or nudemice which are treated with pristane (2,6,10,14-tetramethylpentadecane)by its intraperitoneal administration and reared for 2 weeks, at a doseof 5×10⁶ to 2×10⁷ cells per animal. The hybridoma becomes ascites tumorin 10 to 21 days. The ascitic fluid is collected from the mice or thenude mice and centrifuged, and then an IgG or IgM fraction is recoveredby salting out with 40 to 50% saturated ammonium sulfate or caprylicacid precipitation method, or using a DEAE-Sepharose column, a protein Acolumn, a Cellulofine GSL 2000 (Seikagaku Corp.) column or the like togive a purified monoclonal antibody.

Determination of the subclass of the purified monoclonal antibody can becarried out using a mouse monoclonal antibody typing kit, a ratmonoclonal antibody typing kit or the like. The protein concentrationcan be calculated by the Lowry method or from the absorbance at 280 nm.

The antibody subclass is an isotype within a class, and IgG1, IgG2a,IgG2b and IgG3 can be cited in the case of mouse, and IgG1, IgG2, IgG3and IgG4 in the case of human.

(7) Activity Evaluation of Monoclonal Antibody

Binding activity of a culture supernatant or purified anti-hIGFmonoclonal antibody to hIGF can be measured by the binding ELISAdescribed above 1(5), competitive ELISA, biosensor BIACORE and the like.

The binding ELISA is a method in which binding activity of an antigenand an antibody is measured by immobilizing an antigen on a 96 wellELISA plate, allowing it to react with a primary antibody, allowing alabeled secondary antibody capable of recognizing the primary antibodyto react therewith, and then detecting the label. Examples of theantigen to be immobilized include, purified proteins of hIGF-I andhIGF-II, peptides having partial sequences thereof and the like.Examples of the primary antibody include substances to be measured suchas hybridoma culture supernatants, and purified antibodies. Examples ofthe secondary antibody include antibodies which can recognize theprimary antibody and are labeled with biotin, an enzyme, achemiluminescence substance, a radioisotope or the like. Specifically, ahorseradish peroxidase-labeled anti-rat immunoglobulin (hereinafterreferred to as rIg) mouse antibody and the like can be examplified.

The competitive ELISA is a method in which hIGF-I or hIGF-II isimmobilized in advance on the ELISA plate, an antibody as the substanceto be measured and hIGF-I or hIGF-II are simultaneously added theretoand allowed to react, and the reactivity of another or the same antigenadded to the reaction solution to inhibit the reaction of the antigenimmobilized on the plate with the antibody to be measured is measuredbased on the changes in the amount of the primary antibody binding tothe plate. Changes in the binding amount of the antibody are detected bythe secondary antibody to the antibody. Also, reactivity with a naturaltype hIGF and antigen epitope can be analyzed by the competitive ELISAusing the natural type hIGF and a partial peptide of the hIGF. Whetheror not the antibody is recognizing three-dimensional structure of thehIGF can be examined by a conventional structural analysis. As thestructural analysis, x-ray crystallographic analysis, magnetic nuclearresonance analysis and the like can, for example, be exemplified.

According to the measurement with a biosensor BIACORE, a very smallquantity of change in mass generated on the surface of a sensor tipaccompanied by the association and dissociation between two molecules isdetected as SPR signal by an optical phenomenon. From the associationconstant (hereinafter referred to as Kass) and dissociation constant(hereinafter referred to as Kdiss) obtained from the measurement by thismethod, a binding constant (hereinafter referred to as K_(A)) ofK_(A)=Kass/Kdiss is calculated. K_(A) is expressed by a unit of M⁻¹. Themeasurement with a biosensor BIOCORE can be carried out under optimummeasuring conditions in accordance with the instructions attachedthereto. Regarding the optimum measuring conditions, it is desirablethat amount of the ligand to be immobilized on the sensor tip is withinthe range between the minimum value calculated by formula 1 and themaximum value calculated by formula 2. Also, it is desirable thatbinding amount of the analyte is equal to or smaller than the maximumbinding amount calculated by formula 3. In formulae 1, 2 and 3, ligandmeans a molecule to be immobilized on the sensor tip, analyte means amolecule to be added via a channel system, and S means the number ofligand binding site. RU is abbreviation of resonance unit whichindicates changed amount of mass per unit area on the sensor tipsurface, wherein 1 RU=1 pg/mm². According to the measurement with abiosensor BIACORE, analysis of the binding constant based on the bindingmode of each protein can be carried out by setting flow rate and washingcondition such that the maximum binding amount can be maintained.Minimum immobilized amount (RU)=200×1/S×(molecular weight ofligand/molecular weight of analyte)  Formula 1Maximum immobilized amount (RU)=1000×1/S×(molecular weight ofligand/molecular weight of analyte)  Formula 2Maximum binding amount=molecular weight of analyte×immobilized amount ofligand (RU)/molecular weight of ligand×S  Formula 3

In addition, the activity of the antibody of the present invention toinhibit functions of hIGF can be measured by examining influence of theantibody upon in Vivo or in vitro growth of a cell strain showinghIGF-dependent growth.

The influence upon the growth of a cell strain showing hIGF-dependentgrowth means influence of the antibody of the present invention or anantibody fragment thereof upon Anvil cell growth of a cell strainshowing hIGF-dependent growth, in the presence of hIGF, or upon in vivocell growth of a cell strain showing hIGF-dependent growth, which istransplanted into an animal such as mouse.

As the in vitro cell growth of a cell strain showing hIGF-dependentgrowth in the presence of hIGF, cell growth when a cell is culturedusing a medium prepared by adding hIGF to an hIGF-free basal medium orthe like can be examplified. As the hIGF-free basal medium, TF/BSAmedium [a medium prepared by adding 10 μg/ml of human transferrin(manufactured by Gibco BRL) and 200 μg/ml of BSA to D-MEM/F-12(manufactured by Gibco BRL)] and the like can be exemplified. Regardingthe method for measuring cell growth, it can be measured using a cellgrowth reagent WST-1 (manufactured by Roche).

Regarding the in vivo cell growth of a cell strain showinghIGF-dependent growth, growth of a cell in the animal body when the cellis transplanted into an animal such as mouse can be exemplified.Regarding the method for measuring cell growth, for example, when thecell is developed in the body of a mouse as a tumor mass, it is possibleto measure volume of the tumor mass and use the value as an index of thecell growth.

As the cell strain showing hIGF-dependent growth, a human breast cancercell line MCF7 (ATCC HTB-22), a human colon cancer cell line HT-29 (ATCCHTB-38), a human osteosarcoma cell line MG63 (ATCC CRL-1427) and thelike can be exemplified. In addition, a transformant introduced with anhIGF-I gene can also be exemplified.

Examples of the transformant introduced with an hIGF-I gene include acell strain into which a cloned hIGF-I gene is introduced so thatexpressed amount of hIGF-I is increased in comparison with the case ofnot introducing the hIGF-I gene. Specific examples include atransformant which is prepared by introducing the hIGF-I gene into ahuman lung cancer cell strain A549 cell (ATCC CCL-185). Regarding thehIGF-I gene, the sequence described in a reference (MolecularEndocrinology, 4, 1914-1920, 1990) can be cloned by a method such asPCR.

2. Preparation of Humanized Antibody

(1) Construction of Vector for Expression of Humanized Antibody

As the vector for expression of humanized antibody, it may be anyexpression vector for animal cell use into which a gene coding for CHand/or CL of a human antibody is integrated. The vector for expressionof humanized antibody can be constructed by respectively cloning genescoding for CH and CL of a human antibody into expression vector foranimal cell use.

The C region of a human antibody can be CH and CL of an optional humanantibody, and its examples include a C region of IgG1 subclass of humanantibody H chain (hereinafter referred to as hCγ1), a C region of κclass of human antibody L chain (hereinafter referred to as hCκ) and thelike. As the genes coding for CH and CL of a human antibody, achromosomal DNA comprising exons and introns can be used, and cDNAs canalso be used.

As the expression vector for animal cell use, any vector can be usedwith the proviso that it can integrate and express a gene coding for theC region of a human antibody. For example, pAGE107 (Cytotechnology, 3,133-140, 1990), pAGE103 (Journal of Biochemistry, 101, 1307-1310, 1987),pHSG274 (Gene, 27, 223-232, 1984), pKCR (Proceedings of the NationalAcademy of Sciences of the Untied States of America, 78, 1527-1531,1981), pSG1βd2-4 (Cytotechnology, 4, 173-180, 1990) and the like can beexemplified. As the promoter and enhancer to be used in the expressionvector for animal cell use, SV40 early promoter and enhancer (Journal ofBiochemistry, 101, 1307-1310, 1987), LTR promoter and enhancer ofMoloney mouse leukemia virus (Biochemical & Biophysical ResearchCommunications, 149, 960-968, 1987), promoter (Cell, 41, 479-487, 1985)and enhancer (Cell, 33, 717-728, 1983) of immunoglobulin H chain and thelike can be exemplified.

As the vector for expression of humanized antibody, either of a type inwhich the antibody H chain and L chain are present in different vectorsor a type in which they are present in the same vector (hereinafterreferred to as tandem-type) can be used, a tandem-type vector forexpression of humanized antibody is preferable in view of easiness forconstruction a humanized antibody expression vector, easiness forintroducion into animal cells and easiness for equality of the amount ofthe expressed antibody H chain and L chain in an animal (Journal ofImmunological Methods, 167, 271-278, 1994). Examples of the tandem-typevector for expression of humanized antibody include pKANTEX93 (WO97/10354), pEE18 (Hybridoma, 17, 559-567, 1998) and the like.

The constructed vector for expression of humanized antibody can be usedfor the expression of human chimeric antibodies and human CDR-graftedantibodies in animal cells.

(2) Preparation of cDNAs Coding for Antibody V Region of a Non-HumanAnimal and Analysis of Amino Acid Sequence

cDNAs coding for VH and VL of an antibody of a non-human animal, such asa mouse antibody, for example, are obtained in the following manner.

mRNA is extracted from a hybridoma which produces a mouse antibody orthe like, and then cDNAs are synthsized. The synthesized cDNAs arecloned into a vector such as a phage, plasmid or the like to prepare acDNA library. A recombinant phage or recombinant plasmid having a cDNAcoding for VH or a recombinant phage or recombinant plasmid having acDNA coding for VL are respectively isolated from said library using a Cregion moiety or V region moiety of a mouse antibody as a probe. Thefull length nucleotide sequences for the intended VH and VL of the mouseantibody on the recombinant phage or recombinant plasmid is determined,and the full length amino acid sequences of VH and VL are deduced fromthe nucleotide sequence.

As a non-human animal, any of animals capable of producing a hybridoma,such as mouse, rat, hamster, rabbit and the like.

A guanidine thiocyanate-cesium trifluoroacetate method (Method inEnzymology, 154, 3-28, 1987) can be exemplified as the method forpreparing total RNA from a hybridoma, and an oligo(dT) immobilizedcellulose column method (Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Lab. Press New York, 1989) as the method for preparingmRNA from total RNA. Also, as the kit for preparing mRNA from ahybridoma, Fast Track mRNA Isolation Kit (manufactured by Invitrogen),Quick Prep mRNA Purification Kit (manufactured by Amersham Pharmacia)and the like can be exemplified.

Examples of the methods for synthesizing cDNA and preparing a cDNAlibrary include conventional methods (Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Lab. Press New York, 1989; Current Protocolsin Molecular Biology, Supplement 1-34), or methods using commerciallyavailable kits such as Super Script™ Plasmid System for cDNA Synthesisand Plasmid Cloning (manufactured by GIBCO BRL), ZAP-cDNA Synthesis Kit(manufactured by Stratagene) and TimeSaver cDNA Synthesis Kit(manufactured by Amersham-Pharmacia).

In preparing a cDNA library, as the vector to be used for theintegration of cDNA synthesized using mRNA extracted from a hybridoma asthe template, any vector capable of integrating said cDNA can be used.The examples include phage and plasmid such as ZAP Express (Strategies,5, 58-61, 1992), pBluescript II SK(+) (Nucleic Acid Research, 17, 9494,1989), λZAP II (manufactured by Stratagene), λgt10 and λgt11 (DNACloning: A practical Approach, 1, 49, 1985), Lambda BlueMid(manufactured by Clontech), % λExCell and pT7T318U (manufactured byAmersham-Pharmacia), pcD2 (Molecular & Cellular Biology, 3, 280-289,1983) and pUC18 (Gene, 33, 103-119, 1985).

As the Escherichia coli into which a cDNA library constructed by a phageor plasmid vector is introduced, any strain can be used with the provisothat said cDNA library can be introduced, expressed and maintained. Theexamples include XL1-Blue MRF' (Journal of Biotechnology, 23, 271-289,1992), C600 (Genetics, 59, 177-190, 1968), Y1088 and Y1090 (Science,222, 778-782, 1983), NM522 (Journal of Molecular Biology, 166, 1-19,1983), K802 (Journal of Molecular Biology, 16, 118-133, 1966) and JM105(Gene, 38, 275-276, 1985).

As the method for selecting a cDNA clone coding for VH and VL of anantibody of a non-human animal from a cDNA library, it can be selectedby a colony hybridization method or plaque hybridization method(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. PressNew York, 1989) which uses probe labeled with a radioisotope, afluorescent substance or an enzyme. In addition, a cDNA coding for VHand VL can also be prepared by carrying out the polymerase chainreaction (hereinafter referred to as PCR method; Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Lab. Press New York, 1989; CurrentProtocols in Molecular Biology, Supplement 1-34) by preparing primersand using a cDNA synthesized from mRNA, or a cDNA library, as thetemplate.

Nucleotide sequence of said cDNA can be determined by digesting the cDNAselected by the above method with appropriate restriction enzymes,cloning the digests into a plasmid vector such as pBluescript SK(−)(manufactured by Stratagene), and then carrying out a reaction such as aconventionally used nucleotide sequence analyzing method, for example,the dideoxy method (Proceedings of the National Academy of Sciences ofthe Untied States of America, 74, 5463-5467, 1977) and analyzing theresults using an automatic nucleotide sequence analyzer such as anautomatic nucleotide sequence analyzer ABI PRISM 377 (manufactured byApplied Biosystems) or the like.

By deducing full length amino acid sequence of VH and VL from thedetermined nucleotide sequence and comparing the result with full lengthamino acid sequences of VH and VL of known antibodies (Sequences ofProteins of Immunological Interest, US Dept. Health and Human Services,1991), whether or not the thus obtained cDNA encodes full length aminoacid sequence of VH and VL of the antibody containing secretion signalsequence can be verified. Regarding the full length amino acid sequenceof VH and VL of the antibody containing secretion signal sequence,length of the secretion signal sequence and the N terminal sequence canbe deduced and subgroups to which they belong can be alarified bycomparison with full length amino acid sequences of VH and VL of knownantibodies (Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services, 1991). In addition, amino acid sequence ofeach CDRs of VH and VL can also be found by comparing with amino acidsequences of VH and VL of known antibodies (Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services, 1991).

Also, novelty of the sequences can be examined by carrying out sequencehomology retrieval such as BLAST method (Journal of Molecular Biology,215, 403-410, 1990) on an optional data base, for example, SWISS-PROT,PIR-Protein or the like, using the full length amino acid sequence of VHand VL.

(3) Construction of Human Chimeric Antibody Expression Vector

A human chimeric antibody expression vector can be constructed bycloning cDNAs coding for VH and VL of an antibody of a non-human animalinto upstream of the gene coding for CH and CL of a human antibody inthe vector for expression of humanized antibody described in the above2(1). For example, a human chimeric antibody expression vector can beconstructed by respectively ligating cDNAs coding for VH and VL of anantibody of a non-human animal to a synthetic DNA which comprises3′-terminal side nucleotide sequences of VH and VL of an antibody of anon-human animal and 5′-terminal side nucleotide sequences of CH and CLof a human antibody and also has appropriate restriction enzymerecognizing sequences on both termini, and by respectively cloning theminto upstream of the gene coding for CH and CL of a human antibody inthe vector for expression of humanized antibody described in the above2(1) in such a manner that they are expressed in appropriate forms. Inaddition, a human chimeric antibody expression vector can be constructedby amplifying cDNAs coding for VH and VL of an antibody of a non-humananimal by PCR method using plasmids containing cDNAs coding for the VHand VL as templates and using primers having appropriate restrictionenzyme recognizing sequences on 5′-termini, and respectively cloningthem into upstream of the gene coding for CH and CL of a human antibodyin the vector for expression of humanized antibody described in theabove 2(1) in such a manner that they are expressed in suatable forms.

(4) Construction of cDNAs Coding for V Region of a Human CDR-GraftedAntibody

cDNAs coding for VH and VL of a human CDR-grafted antibody can beconstructed in the following manner. Firstly, FR amino acid sequences ofVH and VL of a human antibody are selected for grafting the intended CDRamino acid sequences of VH and VL of an antibody of a non-human. As theFR amino acid sequences of VH and VL of a human antibody, any sequencescan be used with the proviso that they are derived from a humanantibody. The examples include the FR amino acid sequences of VH and VLof human antibodies registered in data bases such as Protein Data Bankand consensus amino acid sequences of FR VH and VL of subgroups of humanantibodies (Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services, 1991) and the like, but for the purpose ofpreparing a human CDR-grafted antibody having sufficient activity, it isdesirable to select amino acid sequences having a homology of as high aspossible (at least 60% or more) with the intended FR amino acidsequences of VH and VL of an antibody of a non-human animal. Next, aminoacid sequences of VH and VL of a human CDR-grafted antibody are designedby grafting the intended CDR amino acid sequences of VH and VL of anantibody of a non-human animal to the thus selected FR amino acidsequences of VH and VL of a human antibody. The designed amino acidsequences are converted into nucleotide sequences in consideration ofthe codon frequency found in nucleotide sequences of antibody genes(Sequences of Proteins of Immunological Interest, US Dept. Health andHuman Services, 1991). Based on the nucleotide sequences thus designed,several synthetic DNA fragments having a length of about 100 bases aresynthesized, and PCR is carried out using them. In this case, it isprefarable to design six synthesized DNAs for each of VH and VL in viewof the PCR efficiency and the length of DNA to be synthesized.

In addition, DNAs can be easily cloned into the vector for expression ofhumanized antibody constructed in the above 2(1) by introducingappropriate restriction enzyme recognizing sequences into both 5′termini of the synthetic DNA. After the PCR, the amplified products arecloned into plasmids such as pBluescript SK(−) (manufactured byStratagene) and their nucleotide sequences are determined by the methoddescribed in the above 2(2) to obtain plasmids having nucleotidesequences coding for the intended amino acid sequences of VH and VL ofthe human CDR-grafted antibody.

(5) Modification of V Region Amino Acid Sequences of Human CDR-GraftedAntibody

It is known that antigen binding activity of a human CDR-graftedantibody is reduced in comparison with the case of the original antibodyof a non-human animal, when only the desired CDRs of VH and VL of anantibody of a non-human animal are grafted to the FRs of VH and VL of ahuman antibody (BIO/TECHNOLOGY, 9, 266-271, 1991). As the cause of this,it is considered that some amino acid residues of not only CDRs but alsoFRs are involved in the antigen binding activity directly or indirectlyin the VH of VL of the original antibody of a non-human animal,concerned in the antigen binding activity, and these amino acid residuesare considered to be changed to another amino acid residues of the FRsof VH and VL of human antibody according to the grafting of CDRs. Withthe aim of solving this problem, in the case of a human CDR-graftedantibody, an attempt has been made to identify certain amino acidresidues in the amino acid sequences of FRs of VH and VL of the humanantibody, which are directly concerned in the binding with the antigenor which are indirectly concerned in the binding with the antigen byinteracting with amino acid residue of CDR or maintainingthree-dimensional structure of the antibody, and to increase the oncereduced antigen binding activity by modifying them into the amino acidresidues which are found in the original antibody of a non-human animal(BIO/TECHNOLOGY, 9, 266-271, 1991). In preparing a human CDR-graftedantibody, it is the most important point to efficiently identify such FRamino acid residues concerned in the antigen binding activity, andconstruction and analysis of the three-dimensional structure ofantibodies by an X-ray crystallographic analysis (Journal of MolecularBiology, 112,535-542, 1977), a computer modeling (Protein Engineering,7, 1501-1507, 1994) or the like have been carried out for this purpose.Information of the three-dimensional structure of these antibodies haveproduced many useful information for the preparation of humanCDR-grafted antibodies, but on the other hand, a method for preparing ahuman CDR-grafted antibody which is applicable to every antibody has notbeen established yet, and it is necessary at present to carry outvarious try and error efforts, for example by preparing several modifiedbodies from each antibody and examining their correlation to respectiveantigen binding activities.

Modification of FR amino acid residues of VH and VL of a human antibodycan be achieved by carrying out the PCR method described in the above2(4) using a synthetic DNAs for mutagenesis. Nucleotide sequence of theamplified product by PCR is determined by the method described in theabove 2(2) to confirm that the desired modification was attained.

(6) Construction of Human CDR-Grafted Antibody Expression Vector

A human CDR-grafted antibody expression vector can be constructed bycloning cDNAs coding for the VH and VL of human CDR-grafted antibodyconstructed in the above 2(4) and (5) into upstream of the genes codingfor human antibody CH and CL contained in the vector for expression ofhumanized antibody described in the above 2(1).

For example, by introducing appropriate restriction enzyme recognizingsequences into both 5′ termini of the synthetic DNA which is used forconstructing the VH and VL of a human CDR-grafted antibody in the above2(4) and (5), they can be cloned into upstream of the genes coding forCH and CL of a human antibody contained in the vector for expression ofhumanized antibody described in the above 2(1) in such a manner thatthey are expressed in suitable forms.

(7) Transient Expression of Humanized Antibody

In order to efficiently evaluate the antigen binding activity of manytypes of the prepared humanized antibodies, transient expression of thehumanized antibodies can be carried out using the humanized antibodyexpression vectors described in the above 2(3) and (6) or usingexpression vectors mutated. As the host cell into which the expressionvector is introduced, any cell can be used with the proviso that it is ahost cell which can express the humanized antibody, and a monkeykidney-derived cell strain COS-7 cell (ATCC CRL 1651) is generally used(Methods in Nucleic Acids Research, CRC Press, 283, 1991) inconsideration of its large amount of expression. Examples of the methodfor introducing expression vector into COS-7 cell include theDEAE-dextran method (Methods in Nucleic Acids Research, CRC Press, 283,1991), the lipofection method (Proceedings of the National Academy ofSciences of the United States of America, 84, 7413-7417, 1987) and thelike.

After introduction of the expression vector, amount of expression andantigen binding activity of the humanized antibody in the culturesupernatant can be measured by ELISA (Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Chapter 14, 1988; Monoclonal Antibodies:Principles and Practice, Academic Press Limited, 1996) and the like.

(8) Stable Expression of Humanized Antibody

A transformant capable of stably expressing a humanized antibody can beobtained by introducing the humanized antibody expression vectordescribed in the above 2(3) or (6) into an appropriate host cell.

As the method for introducing the expression vector into a host cell,the electroporation method (Cytotechnology, 3, 133-140, 1990) and thelike can be exemplified.

As the host cell into which the humanized antibody expression vector isintroduced, any cell can be used with the proviso, that it is a hostcell which can express the humanized antibody, and the examples includemouse SP2/0-Ag14 cell (ATCC CRL 1581), mouse P3X63-Ag8.653 cell (ATCCCRL 1580), a dihydrofolate reductase gene (hereinafter referred to asdhfr)-deficient CHO cell (Proceedings of the National Academy ofSciences of the United States of America, 77, 4216-4220, 1980), ratYB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL 1662, hereinafter referred to asYB2/0 cell) and the like.

After introduction of the expression vector, the obtained transformantis cultured using a medium for animal cell containing drugs such as G418sulfate (hereinafter referred to as G418) in accordance with the methoddisclosed in Japanese published unexamined application No. 257891/90,and a transformant which stably expresses the humanized antibody can beselected. Examples of the medium for animal cell includes RPMI 1640medium (manufactured by Nissui Pharmaceutical), GIT medium (manufacturedby Nihon Seiyaku), EX-CELL 302 medium (manufactured by JRH), IMDM(manufactured by GIBCO BRL) and Hybridoma-SFM (manufactured by GIBCOBRL), or media prepared by adding various additives such as FCS to thesemedia. The obtained transformant is cultured in a medium, and thehumanized antibody can be expressed and accumulated in a culturesupernatant. The amount of expression and antigen binding activity ofthe humanized antibody in the culture supernatant can be measured byELISA. In addition, amount of expression of the humanized antibody bythe transformant can be increased by utilizing of a dhfr amplificationsystem or the like in accordance with the method disclosed in Japanesepublished unexamined application No. 257891/90.

The humanized antibody can be purified from the culture supernatant oftransformant cell using a protein A column (Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Chapter 8, 1988; MonoclonalAntibodies: Principles and Practice, Academic Press Limited, 1996). Inaddition, purification methods generally used for the purification ofprotein can also be used. For example, the purification can be carriedout by employing a combination of gel filtration, ion exchangechromatography, ultrafiltration and the like. Molecular weight of the Hchain, L chain or the whole antibody molecule of the purified humanizedantibody can be measured by polyacrylamide gel electrophoresis(hereinafter referred to as PAGE: Nature, 227, 680-685, 1970), westernblotting method (Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Chapter 12, 1988; Monoclonal Antibodies: Principles andPractice, Academic Press Limited, 1996) and the like.

(9) Activity Evaluation of Humanized Antibody

Binding activity of anti-IGF humanized antibody in a culture supernatantor purified anti-hIGF humanized antibody to hIGF can be measured by theELISA, biosensor BIACORE or the like shown in the above 1(7). Inaddition, the activity of the antibody of the present invention toinhibit functions of hIGF can be measured by examining influence of theantibody upon in Vivo or in vitro growth of cell strains showinghIGF-dependent growth, as shown in the above 1(7).

3. Preparation of Antibody Fragments

The antibody fragments can be prepared from the anti-hIGF antibodiesdescribed in the above 1 and 2. by genetic engineering techniques orprotein chemistry techniques based.

As the genetic engineering techniques, mentioned are a method in which agene coding for the antibody fragment of interest is constructed, andexpression and purification is carried out using an appropriate hostsuch as an animal cell, plant cell, insect cell, and Escherichia coli.

As the protein chemistry techniques, mentioned are methods includingsite-specific digestion using protease such as pepsin and papain andpurification.

As the antibody fragments, Fab, F(ab′)₂, Fab′ scFv, diabody, dsFv, aCDR-containing peptide and the like can be exemplified.

(1) Preparation of Fab

According to a protein chemistry techniques, Fab can be prepared bytreating IgG with a protease papain. After the papain treatment, whenthe original antibody is an IgG subclass having protein A bindingability, uniform Fab can be recovered by separating it from IgGmolecules and Fc fragments by passing through a protein A column(Monoclonal Antibodies: Principles and Practice, third edition, 1995).In the case of an antibody of IgG subclass having no protein A bindingability, Fab can be recovered by an ion exchange chromatography in afraction eluted with a low salt concentration (Monoclonal Antibodies:Principles and Practice, third edition, 1995). In addition, Fab can alsobe prepared by genetic engineering techniques using Escherichia coli inmost cases or using an insect cell, animal cell or the like. Forexample, an Fab expression vector can be prepared by cloning the DNAcoding for antibody V region obtained in the above 2(2), 2(4) or 2(5)into a vector for expression of Fab. As the vector for expression ofFab, any vector can be used with the proviso that it can effectintergration and expression of a DNA of Fab. For example, pIT106(Science, 240, 1041-1043, 1988) and the like can be examplified. Fab canbe produced and accumulated in an inclusion body or periplasmic space byintroducing the Fab expression vector into an appropriate Escherichiacoli. Active Fab can be obtained from the inclusion body by a refoldingmethod commonly used for protein, and when it is expressed in theperiplasmic space, active Fab is leaked in the culture supernatant.Uniform Fab can be purified after the refolding or from the culturesupernatant using a column immobilized with an antigen (AntibodyEngineering, A Practical Guide, W.H. Freeman and Company, 1992).

(2) Preparation of F(ab′)₂

According to a protein chemistry techniques, F (ab′)₂ can be prepared bytreating IgG with a protease pepsin. After the pepsin treatment, it canbe recovered as uniform F(ab′)₂ by carrying out a purification proceduresimilar to the case of Fab (Monoclonal Antibodies: Principles andPractice, third edition, Academic Press, 1995). In addition, F(ab′)₂ canalso be prepared by the method described in the following 3(3) in whichFab′ is treated with maleimide such as o-PDM and bismaleimide hexane toeffect thioether bonding, or a method in which it is treated with DTNB[5,5′-dithiobis (2-nitrobenzoic acid)] to effect S—S bonding (AntibodyEngineering, A Practical Approach, IRL PRESS, 1996).

(3) Preparation of Fab′

Fab′ can be obtained by treating the F(ab′)₂ described in above 3(2)with a reducing agent such as dithiothreitol. Also, Fab′ can be preparedby genetic engineering techniques using Escherichia coli in most casesor using an insect cell, animal cell or the like. For example, an Fab′expression vector can be prepared by cloning the DNA coding for antibodyV region obtained in the above 2(2), 2(4) or 2(5) into a vector forexpression of Fab′. As the vector for expression of Fab′, any vector canbe used with the proviso that it can effect intergration and expressionof a DNA for Fab′ use. For example, pAK19 (BIO/TECHNOLOGY, 10, 163-167,1992) and the like can be examplified. Fab′ can be produced andaccumulated in an inclusion body or periplasmic space by introducing theFab′ expression vector into an appropriate Escherichia coli. Active Fab′can be obtained from the inclusion body by a refolding method commonlyused for protein, and when it is expressed in the periplasmic space, itcan be recovered into extracellulary by disrupting the cells by atreatment such as lysozyme partial digestion, osmotic shock and ultrasonication. Uniform Fab′ can be purified after the refolding or from thedisrupted cell suspension using a protein G column or the like (AntibodyEngineering, A Practical Approach, IRL PRESS, 1996).

(4) Preparation of scFv

According to genetic engineering techniques, scFv can be prepared usinga phage or Escherichia coli, or an insect cell, animal cell or the like.For example, an scFv expression vector can be prepared by cloning DNAscoding for the V region of the antibody described in the above 2(2),2(4) or 2(5) into a vector for expression of scFv. As the vector forexpression of scFv, any vector can be used with the proviso that it caneffect intergration and expression of a DNA of scFv. For example,pCANTAB5E (manufactured by Amersham-Pharmacia), pHFA (Human Antibodies &Hybridomas, 5, 48-56, 1994) and the like can be examplified. Byintroducing the scFv expression vector into an appropriate Escherichiacoli and infecting the cells with a helper phage, a phage whichexpresses scFv on the phage surface in a fused form with the phagesurface protein can be obtained. Also, scFv can be produced andaccumulated in the inclusion body or periplasmic space of scFvexpression vector-introduced Escherichia coli. Active scFv can beobtained from the inclusion body by a refolding method commonly used forprotein, and when it is expressed in the periplasmic space, it can berecovered into extracellulary by disrupting the cells by a treatmentsuch as lysozyme partial digestion, osmotic shock and ultra sonication.Uniform scFv can be purified after the refolding or from the disruptedcell suspension using a cation exchange chromatography or the like(Antibody Engineering, A Practical Approach, IRL PRESS, 1996).

(5) Preparation of Diabody

By genetic engineering techniques, diabody can be prepared usingEscherichia coli in most cases or using an insect cell, animal cell orthe like. For example, a diabody expression vector can be prepared bypreparing DNAs coding for the VH and VL of the antibody described in theabove 2(2), 2(4) or 2(5) are linked to each other in such a manner thatthe number of amino acid residues encoded by the linker becomes 8residues or less, and cloning it into a vector for expression ofdiabody. As the vector for expression of diabody, any vector can be usedwith the proviso that it can effect intergration and expression of a DNAof diabody. For example, pCANTAB5E (manufactured. byAmersham-Pharmacia), pHFA (Human Antibodies Hybridomas, 5, 48, 1994) andthe like can be examplified. Diabody can be produced and accumulated inthe inclusion body or periplasmic space of diabody expressionvector-introduced Escherichia coli. Active diabody can be obtained fromthe inclusion body by a refolding method commonly used for protein, andwhen it is expressed in the periplasmic space, it can be recovered intoextracellulary by disrupting the cells by a treatment such as lysozymepartial digestion, osmotic shock and ultra sonication. Uniform scFv canbe purified after the refolding or from the disrupted cell suspensionusing a cation exchange chromatography or the like (AntibodyEngineering, A Practical Approach, IRL PRESS, 1996).

(6) Preparation of dsFv

According to genetic engineering techniques, dsFv can be prepared usingEscherichia coli in most cases, or using an insect cell, animal cell orthe like. Firstly, DNAs in which an encoded amino acid residue isreplaced by cysteine are prepared by introducing mutation intoappropriate positions of the DNAs coding for antibody VH and VLdescribed in the above 2(2), 2(4) or 2(5). VH and VL expression vectorscan be prepared by cloning each of the DNA thus prepared into a vectorfor expression of dsFv. AS the vector for expression of dsFv, any vectorcan be used with the proviso that it can effect intergration andexpression of a DNA of dsFv. For example, pULI9 (Protein Engineering, 7,697-704, 1994) and the like can be examplified. By introducing the VHand VL expression vectors into an appropriate Escherichia coli, they canbe produced and accumulated in the inclusion body or periplasmic space.By obtaining the VH and VL from the inclusion body or periplasmic spaceand mixing them, active dsFv can be obtained by a refolding methodcommonly used for protein. After the refolding, it can be furtherpurified by an ion exchange chromatography, gel filtration and the like(Protein Engineering, 7, 697-704, 1994).

(7) Preparation of CDR Peptide

A peptide containing CDR can be prepared by chemical synthesis methodsuch as Fmoc method and tBoc method. A CDR peptide expression vector canbe prepared by preparing a DNA coding for a CDR-containing peptide, andcloning the DNA thus prepared into an appropriate vector for expression.As the vector for expression, any vector can be used with the provisothat it can effect integration and expression of a DNA coding forCDR-containing peptide. For example, pLEX (manufactured by Invitrogen),pAX4a+ (manufactured by Invitrogen) and the like can be examplified. Byintroducing the expression vector into an appropriate Escherichia coli,the peptide can be produced and accumulated in the inclusion body orperiplasmic space. By obtaining the CDR peptide from the inclusion bodyor periplasmic space, it can be purified by an ion exchangechromatography, gel filtration and the like (Protein Engineering, 7,697-704, 1994).

(8) Activity Evaluation of Antibody Fragments

Binding activity of the purified antibody fragments to hIGF can bemeasured by the ELISA, biosensor BIACORE and the like shown in the above1(7). In addition, the activity of the antibody of the invention toinhibit functions of hIGF can be measured by examining influence of theantibody upon in vivo or in vitro growth of the cell strains showinghIGF-dependent growth, as shown in the above 1(7).

4. Methods for Detecting and Determining hIGF Using Anti-hIGF Antibody

The present invention relates to methods for immunologically detectingand determining hIGF using the antibody of the present invention.Accordingly, the antibody of the present invention can be used for thediagnosis of hIGF-mediated diseases and diseases showing pathologicalprogressing due to abnormally accelerated hIGF production, which will bedescribed later.

Regarding the method for immunologically detecting or determining hIGFusing the antibody of the present invention, fluorescent antibodytechnique, ELISA, radioimmunoassay (hereinafter referred to as RIA),immunohistochemical staining methods such as immune tissue stainingmethod and immunocyte staining method (ABC method, CSA method and thelike), sandwich ELISA (Monoclonal Antibody Experimentation Manual,Kodansha Scientific, 1987; Second Series Biochemistry ExperimentationCourse 5, Immunobiochemistry studies, Tokyo Kagaku Dojin, 1986) and thelike can be exemplified.

The fluorescent antibody technique is a method in which the antibody ofthe present invention is allowed to react with an isolated cell ortissue and further allowed to react with an anti-Ig antibody or antibodyfragment labeled with a fluorescent substance such as fluoresceinisothiocyanate (hereinafter referred to as FITC) and then thefluorescence dye is measured using a flow cytometer.

The RIA is a method in which the antibody of the present invention isallowed to react with a cell or disrupted solution thereof, a tissue ordisrupted solution thereof, a cell culture supernatant, serum, pleuraleffusion, ascites, ophthalmic fluid or the like and further allowed toreact with an anti-Ig antibody or antibody fragment treated with aradioisotope labeling and then the isotope is measured using ascintillation counter or the like.

The immunocyte staining method or immune tissue staining method is amethod in which the antibody of the present invention is allowed toreact with an isolated cell or tissue and further allowed to react withan anti-Ig antibody or antibody fragment treated with a fluorescentmaterial such as FITC and an enzyme label such as peroxidase and biotinand then the sample is observed under a microscope.

The sandwich ELISA is a method in which one of two antibodies of thepresent invention having different antigen recognizing sites first isimmobilized to an ELISA plate, the other antibody is labeled with afluorescent substance such as FITC or an enzyme such as peroxidase andbiotin, the antibody-immobilized plate is allowed to react with anisolated cell or disrupted solution thereof, a tissue or disruptedsolution thereof, a cell culture supernatant, serum, pleural effusion,ascites, ophthalmic fluid or the like, and then the labeled antibody isallowed to react therewith and a reaction corresponding to each label iscarried out.

5. Diagnosis and Treatment of hIGF-Mediated Diseases and DiseasesShowing Pathological Progressing Due to Abnormally Promoted hIGFProduction

Since the anti-hIGF antibody of the present invention and antibodyfragments thereof specifically bind to hIGF-I and hIGF-II and inhibittheir functions, it is considered that they are useful for diagnosingand treating hIGF-mediated diseases and diseases showing pathologicalprogressing due to abnormally promoted hIGF production. In addition mostpart of a humanized antibody is derived from an amino acid sequence of ahuman antibody in comparison with an antibody of a non-human animal, itdoes not show immunogenicity in the human body, and its repeatedadministration is possible and long-term persistency of its effect isexpected.

As the method for diagnosing hIGF-mediated diseases and diseases showingpathological progressing due to abnormally promoted hIGF production, themethod described in the above 4 in which hIGF existing in a biologicalsample of a person to be tested such as cell, tissue and serum isimmunologically detected can be exemplified.

The anti-hIGF antibody of the present invention or an antibody fragmentthereof can be administered as it is, but it is desirable in general toprovide it as a pharmaceutical preparation produced by an optionalmethod well known in the technical field of manufacturing pharmacy, bymixing it with one or more pharmacologically acceptable carriers.

As the administration route, it is advisable to use the most effectiveroute in the treatment. Examples thereof can include oral administrationand parenteral administrations such as intraoral, intratracheal,intrarectal, subcutaneous, intramuscular, intraarticular and intravenousadministrations. In case of the antibody or peptide preparations,intraarticular and intravenous administrations are preferable.

Examples of the administration form include sprays, capsules, tablets,granules, syrups, emulsions, suppositories, injections, ointments, tapesand the like.

Examples of appropriate preparations for oral administration includeemulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups can be produced byusing, as additives, water, saccharides such as sucrose, sorbitol andfructose, glycols such as polyethylene glycol and propylene glycol, oilssuch as sesame oil, olive oil and soybean oil, antiseptics such asp-hydroxybenzoic acid esters, and flavors such as strawberry flavor andpeppermint.

Capsules, tablets, powders, granules and the like can be produced byusing, as additives, excipients such as lactose, glucose, sucrose andmannitol, disintegrating agents such as starch and sodium alginate,lubricants such as magnesium stearate and talc, binders such aspolyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants suchas fatty acid esters, and plasticizers such as glycerin.

Examples of preparations appropriate for parenteral administrationinclude injections, suppositories, sprays and the like.

Injections are prepared by using a carrier comprising a salt solution, aglucose solution or a mixture of both, and the like.

Suppositories are prepared using a carrier such as cacao butter,hydrogenated fat or carboxylic acid.

Sprays are prepared by using the antibody or the peptide as such or incombination with a carrier which facilitates dispersion and absorptionof the antibody or the peptide in the form of fine particles withoutstimulating the mouth and the airway mucous membrane of a recipient.

Specific examples of the carrier include lactose, glycerin and the like.Preparations such as aerosol and dry powder can be formed depending onproperties of the antibody or the peptide and the carrier used. Theseparenteral preparations may comprise the ingredients listed as additivesin the oral preparations.

The dose or the number of administrations varies with the desiredtherapeutic effects, the administration method, the therapeutic period,the age, the body weight and the like. It is usually from 10 μg/kg to 10mg/kg per day for an adult.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows reactivity of anti-hIGF rat monoclonal antibody specificfor hIGF-I (binding ELISA).

FIG. 2 shows reactivity of anti-hIGF rat monoclonal antibody for hIGF-Ihaving natural three-dimensional structure in a liquid system(competitive ELISA).

FIG. 3 shows activity of various peptides to inhibit binding ofanti-hIGF rat monoclonal antibody KM1468 to hIGF-I. The abscissa showsconcentration of respective peptides (μg/ml), and the ordinate showsbinding activity (%). The various peptides used are shown in thedrawing.

FIG. 4 shows activities of hIGF-I, hIGF-II and human insulin to inhibitbinding of anti-hIGF antibody KM1468 to hIGF-I and hIGF-II. A showsinhibition by respective factors upon binding of KM1468 to hIGF-I, and Bupon binding of KM1468 to hIGF-II. The abscissa shows concentration ofrespective factors (μg/ml), and the ordinate shows binding activity (%)wherein the value with no addition of factors is defined as 100%. ▪shows the reactivity when hIGF-I was added, and ◯ shows the reactivitywhen hIGF-II was added and Δ shows the reactivity when human insulin wasadded.

FIG. 5 shows influence of anti-hIGF antibody KM1468, sm1.2 and S1F2 uponthe growth of a human breast cancer cell strain MCF7 by hIGF and humaninsulin. A shows cell growth activity by respective factors. Theabscissa shows concentration of respective factors (μg/ml), and theordinate shows growth (OD450). ◯ shows activity of hIGF-I, ▪ showsactivity of hIGF-II and □ shows activity of human insulin. B, C and Dshow influence of respective antibodies upon growth activity by hIGF-I,and by hIGF-II and by human insulin, respectively. The abscissa showsantibody concentration (μg/ml), and the ordinate shows growth (OD450).Fine dotted line shows growth with no addition of antibodies, and dottedline shows growth with no addition of respective factors. ◯ shows theactivity of KM1468, □ shows the activity of sm1.2 and ▪ shows theactivity of S1F2.

FIG. 6 shows influence of anti-hIGF antibody KM1468, sm1.2 and S1F2 uponthe growth of a human colon cancer cell strain HT-29 by hIGF and humaninsulin. A shows cell growth activity by respective factors. Theabscissa shows concentration of respective factors (ng/ml), and theordinate shows growth (OD450). ◯ shows activity of hIGF-I, ● shows theactivity of hIGF-II and □ shows the activity of human insulin. B, C andD show influence of respective antibodies upon growth activity byhIGF-I, by hIGF-II by human insulin, respectively. The abscissa showsantibody concentration (μg/ml), and the ordinate shows growth (OD450).Fine dotted line shows growth with no addition of antibodies, and dottedline shows growth with no addition of respective factors. ◯ shows theactivity of KM1468, open square shows the activity of sm1.2 and ● showsthe activity of S1F2.

FIG. 7 shows influence of anti-hIGF antibody KM1468, sm1.2 and S1F2 uponthe growth of a human osteosarcoma cell strain MG63 by hIGF and humaninsulin. A shows cell growth activity by respective factors. Theabscissa shows concentration of respective factors (ng/ml), and theordinate shows growth (OD450). ◯ shows activity of hIGF-I, ● shows theactivity of hIGF-II and □ shows the activity of human insulin. B, C andD show influence of respective antibodies upon growth activity byhIGF-I, by hIGF-II and by of human insulin, respectively. The abscissashows antibody concentration (μg/ml), and the ordinate shows growth(OD450). Fine dotted line shows growth with no addition of antibodies,and dotted line shows growth with no addition of respective factors. ◯shows the activity of KM1468, □ shows the activity of sm1.2 and ▪ showsthe activity of S1F2.

FIG. 8 is a graph showing construction steps of plasmidsPBS(II)SK(−)/hIGF-I and pKANTEX93/hIGF-I.

FIG. 9 is a drawing showing expression of hIGF-I in A549/hIGF-I cell. Ashows inhibition by a recombinant hIGF-I protein. The abscissa showsconcentration of the added recombinant hIGF-I protein, and the ordinateshows color development (OD415). B shows hIGF-I contained in culturesupernatants of A549 cell and A549/hIGF-I cell. Void shows A549 cell andnetting shows A549/hIGF-I cell.

FIG. 10 shows cell growth inhibitory effect of KM1468 upon hIGF-Iexpressing cells. Broken like shows growth of A549/hIGF-I cell in theabsence of anti-hIGF antibody KM1468, and solid line shows growth ofA549 cell in the absence of anti-hIGF antibody KM1468. ▪ shows growth ofA549/hIGF-I cell in the presence of anti-hIGF antibody KM1468, and ◯shows growth of A549 cell in the presence of anti-hIGF antibody KM1468.

FIG. 11 is a graph showing anchorage independent growth inhibitoryeffect of KM1468. In the drawing, voided column shows the number offormed colonies of A549 cell, netted column shows the number of formedA549/hIGF-I cells, and black-finished column shows the number of formedA549/hIGF-I cells in the presence of anti-hIGF antibody KM1468.

FIG. 12 is a graph showing antitumor effect of anti-hIGF antibodyKM1468. The abscissa shows the number of elapsed days after tumortransplantation, and the ordinate shows tumor volume. Among the micetransplanted with A549 cell, ● shows effect in the absence of anti-hIGFantibody KM1468, and ◯ shows effect in the presence of anti-hIGFantibody KM1468. Among the mice transplanted with A549/hIGF-I cell, ▪shows effect in the absence of anti-hIGF antibody KM1468, and ▪ showseffect in the presence of anti-hIGF antibody KM1468.

FIG. 13 is a drawing showing construction steps of plasmids pKM1468VHand pKM1468VL.

FIG. 14 is a drawing showing construction steps of plasmidpKANTEX1468Chi.

FIG. 15 shows SDS-PAGE (using a 4 to 15% gradient gel) electrophoresispattern of purified anti-hIGF chimeric antibody KM3002. The left side isthe electrophoresis pattern under non-reducing condition, and the rightside is the electrophoresis pattern under reducing condition. Lane Mshows high molecular weight markers under non-reducing condition or lowmolecular weight markers under reducing condition, and lane 1 showselectrophoresis pattern of KM3002.

FIG. 16 shows reaction of anti-hIGF rat antibody KM1468 and anti-hIGFchimeric antibody KM3002 upon hIGF-I. The abscissa shows antibodyconcentration (μg/ml), and the ordinate shows binding activity (OD415).◯ shows reactivity of KM1468, and ● shows reactivity of KM3002.

FIG. 17 shows influence of anti-hIGF antibody KM1468, sm1.2, S1F2 andanti-hIGF chimeric antibody KM3002 upon the growth of a human coloncancer cell strain HT-29 by hIGF. A and B show influence of respectiveantibodies upon growth activity by hIGF-I and by hIGF-II, Δrespectively. The abscissa shows antibody concentration (μg/ml), and theordinate shows growth (OD450). Fine dotted line shows growth in theabsence of antibodies, and dotted line shows growth in the absence ofrespective factors. ◯ shows the activity of KM1468, ● shows the activityof KM3002, Δ shows the activity of sm1.2 and ▴ shows the activity ofS1F2.

The present invention will be described below by referring to examples,but the present invention is not limited thereto.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1

Preparation of Antibody to hIGF-I

(1) Immunization of Animal and Preparation of Antibody Producing Cell

A recombinant hIGF-I (manufactured by R & D) was conjugated withmethylated BSA (manufactured by SIGMA) for the purpose of increasing itsimmunogenicity, and use as the immunogen. That is, the methylated BSAdissolved in redistilled water was mixed at a ratio of methylated BSA:hIGF-I=1:4 (weight ratio) at 4° C. and stirred for 10 seconds using aVortex mixer. Thereafter, this was mixed with Freund's complete adjuvantor Freund's incomplete adjuvant at a volume ratio of 1:1 using a syringeequipped with connecting needles and used as the immunogen (hereinafterreferred to as methylated BSA-hIGF-I adjuvant).

The methylated BSA-hIGF-I adjuvant prepared as described in the aboveusing Freund's complete adjuvant (equivalent, to 100 μg of hIGF-I) wasadministered to an SD rat of 5-weeks-old, and the immunogen prepared inthe same manner using Freund's incomplete adjuvant was administeredstarting 2 weeks thereafter once a week for 4 times in total.

A blood sample was collected from the venous plexus of the fundus of theeye, antibody titer in the serum was examined by the binding ELISA shownin Example 1(4), and the spleen was excised from a rat whose serumshowed a sufficient antibody titer 3 days after the final immunization.

The spleen was cut into pieces in MEM medium (manufactured by NissuiPharmaceutical), unbound using a pair of forceps and centrifuged (1,200rpm, 5 minutes), and then the supernatant was discarded, the resultingprecipitate was treated with Tris-ammonium chloride buffer (pH 7.65) for1 to 2 minutes to eliminate erythrocytes, and the remaining cells werewashed three times with MEM and submitted for cell fusion.

(2) Preparation of Mouse Myeloma Cells

An 8-azaguanine-resistant mouse myeloma cell strain P3-U1 was culturedusing a normal medium, and 2×10⁷ or more of the cells were prepared forthe cell fusion.

(3) Preparation of Hybridoma

The rat spleen cells obtained in Example 1(1) and the myeloma cellsobtained in (2) were mixed at a ratio of 10:1 and centrifuged (1,200rpm, 5 minutes), the supernatant was discarded, and then, while stirringat 37° C., to the precipitated cells were added a fusion medium (amixture composed of 2 g of PEG-1000, 2 ml of MEM and 0.7 ml of dimethylsulfoxide) in an amount of 0.2 to 1.0 ml per 1.0×10² rat spleen cells, 1to 2 ml of MEM-several times at 1- to 2-minute intervals and then aportion of MEM further added thereto to adjust the total volume to 50ml. After centrifugation (900 rpm, 5 minutes), the supernatant wasdiscarded, and the resulting cells were gently loosened and suspended in100 ml of HAT medium {a medium prepared by supplementing the normalmedium [a medium prepared by adding 1.5 mM glutamine, 50 μM2-mercaptoethanol, 10 μg/ml gentamicin and 10% fetal calf serum(hereinafter referred to as FCS) to RPMI-1640 medium] with 0.1 mMhypoxanthine, 15 μM thymidine and 0.4 μM aminopterin}.

This suspension was dispensed in 100 μl/well portions into a 96 wellculture plate and incubated in a 5% CO₂ incubator at 37° C. for a periodof from 7 to 14 days. Wells in which the culture supernatants whichreacted with the methylated BSA-hIGF-I but did not react with a negativecontrol methylated BSA-BSA [a conjugate prepared by carrying out thesame reaction of the above Example 1(1) using BSA] were selected by thebinding ELISA as described in Example 1(4), and anti-hIGF-I ratmonoclonal antibody producing hybridomas were established by carryingout single cell cloning twice by changing the medium to HT medium (amedium prepared by removing aminopterin from the HAT medium) and thenormal medium.

As a result, 6 hybridoma clones KM1468, KM1469, KM1470, KM1471, KM1472and KM1473 having the reactivity shown in FIG. 1 were obtained. Whensubclass of the antibody produced by each hybridoma was examined byELISA using a subclass typing kit, subclass of each antibody was IgG2b.

(4) Selection of Monoclonal Antibody (binding ELISA)

The methylated BSA-hIGF-I prepared in Example 1(1) and the methylatedBSA-BSA as a negative control were used as the antigens to beimmobilized on the ELISA plate. Each of the antigens was dispensed in 50μl/well portions as a BSA concentration of 10 μg/ml into a 96 well ELISAplate (manufactured by Greiner) and allowed to stand overnight at 4° C.to effect its immobilized. After washing with PBS, PBS containing 1% BSA(hereinafter referred to as BSA-PBS) was added in 100 μl/well portionsand allowed to react at room temperature for 1 hour to carry outblocking of the remaining active groups. After discarding BSA-PBS,immunized rat antiserum, culture supernatant of each anti-hIGF-Imonoclonal antibody producing hybridoma or purified anti-hIGF-I ratmonoclonal antibody was dispensed in 50 μl/well portions and allowed toreact at room temperature for 2 hours. After the reaction, each well waswashed with PBS containing 0.05% Tween 20 (hereinafter referred to asTween-PBS), and then 4,000 times-diluted peroxidase-labeled rabbitanti-rat Ig antibody (manufactured by DAKO) was added as the secondaryantibody in 50 μl/well portions and allowed to react at room temperaturefor 1 hour. After the reaction and subsequent washing with Tween-PBS, anABTS substrate solution [a solution prepared by dissolving 0.55 g of2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt in 1liter of 0.1 M citrate buffer (pH 4.2), and further adding thereto 1μl/ml of hydrogen peroxide just before use] was added in 50 μl/wellportions to effect color development, and then absorbance at 415 nm(hereinafter referred to as OD415) was measured using a plate readerEmax (manufactured by Molecular Devices).

(5) Purification of Monoclonal Antibody

Each of the hybridoma-clones obtained in Example 1(3) wasintraperitoneally injected into 8-week-old female Balb/c nude mice whichhad been treated with pristane, at a dose of to 20×10⁶ cells per animal.After 10 to 21 days, the ascitic fluid was collected (1 to 8 ml/animal)from the mice in which the hybridoma change into ascites tumor and thencentrifuged (3,000 rpm, 5 minutes) to remove solid. Thereafter, the IgGfraction was purified by the caprylic acid precipitation method(Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988)and used as a purified monoclonal antibody.

Example 2

Examination of Reactivity of Anti-hIGF-I Rat Monoclonal Antibody

(1) Reactivity to Natural Three-Dimensional Structure of hIGF-I

Reactivity of the anti-hIGF-I rat monoclonal antibodies selected inExample 1(3) with hIGF-I which maintains natural three-dimensionalstructure in a liquid phase system was examined by a competitive ELISAshown below.

Each of 5-fold serial dilutions of hIGF-I starting from 20 μg/ml wasdispensed in 50 μg/well portions into the plate shown in Example 1(4) inwhich the methylated BSA-hIGF-I prepared in Example 1(1) had beenimmobilized, and then each of the solutions prepared by dilutingpurified antibodies of the anti-hIGF-I rat monoclonal antibodies(KM1468: 6.0 μg/ml, KM1470: 1.0 μg/ml, KM1471: 0.16 μg/ml, KM1472: 7.0μg/ml, KM1473: 1.2 μg/ml) was dispensed in 50 μl/well portions, mixedand allowed to react at room temperature for 2 hours. After the reactionand subsequent washing with Tween-PBS, 4,000 times-dilutedperoxidase-labeled rabbit anti-rat Ig antibody (manufactured by DAKO)was added in 50 μl/well portions and allowed to react at roomtemperature for 1 hour. After the reaction and subsequent washing withTween-PBS, the ABTS substrate solution [a solution prepared bydissolving 0.55 g of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonicacid) ammonium salt in 1 liter of 0.1 M citrate buffer (pH 4.2), andfurther adding thereto 1 μl/ml of hydrogen peroxide just before use] wasadded in 50 μl/well portions to effect color development, and then OD415was measured using a plate reader Emax (manufactured by MolecularDevices).

As shown in FIG. 2, each of the anti-hIGF-I rat monoclonal antibodiesshowed the reactivity with natural three-dimensional structure of hIGF-Iin the liquid phase. In addition, in the case of KM1468 which showed thehighest sensitivity, it was able to detect the hIGF-I having naturalthree-dimensional structure contained in the liquid phase system, up toa concentration of 16 ng/ml.

(2) Reactivity of Anti-hIGF Antibody KM1468 with hIGF-I by CompetitiveELISA

A possibility was suggested in Example 2(1) that the anti-hIGF antibodyKM1468 recognizes three-dimensional structure of hIGF-I. However, sincethere is also a possibility that KM1468 recognizes amino acid primarysequence, its reactivity with hIGF-I partial peptides was analyzed.

(2-1) Synthesis of hIGF-I Partial Peptides

Partial peptides of hIGF-I were synthesized in accordance with themethod described in WO 01/64754. The synthesized peptides were peptidescorresponding to a sequence of 1st to 18th positions of hIGF-I (SEQ IDNO: 17, hereinafter referred to as p1-18), 14th to 30th positionsthereof (SEQ ID NO: 18, hereinafter referred to as p14-30), 24th to 35thpositions thereof (SEQ ID NO: 19, hereinafter referred to as p24-35),29th to 41st positions thereof (SEQ ID NO: 20, hereinafter referred toas p29-41), 36th to 47th positions thereof (SEQ ID NO: 21, hereinafterreferred to as p36-47), 41st to 56th positions thereof (SEQ ID NO: 22,hereinafter referred to as p41-56), 52nd to 70th positions thereof (SEQID NO: 23, hereinafter referred to as p52-70), 53rd to 61st positionsthereof (SEQ ID NO: 24, hereinafter referred to as p53-61) and 61st to70th positions thereof (SEQ ID NO: 25, hereinafter referred to asp61-70), and they were designed such that they covered full length ofhIGF-I. Regarding Cys existing in inner part of these peptides,sequences in which it was replaced by Ser or Ala were synthesized. Inaddition, regarding the sequence corresponding to 41st to 56thpositions, a sequence having Cys inside therein (SEQ ID NO: 26,hereinafter referred to as p41-56C) was also synthesized.

(2-2) Analysis of Antigen Recognition Site of Anti-hIGF Antibody KM1468

Analysis of antigen recognition site of anti-hIGF antibody KM1468 wascarried out using the various peptides synthesized in the above (2-1) bya competitive ELISA shown below.

Plates on which antigens were immobilized were prepared as shown inExample 1(4), anti-hIGF antibody KM1468 diluted to 4.0 μg/ml wasdispensed therein in 50 μl/well portions, and then solutions of 3-foldserial dilutions of each peptide prepared by starting from 50 μg/ml,alone or in various combinations, or of hIGF-I were dispensed in 50μl/well portions, mixed and allowed to react at room temperature for 1hour. After the reaction and subsequent washing with Tween-PBS, 4,000times-diluted peroxidase-labeled rabbit anti-rat Ig antibody(manufactured by DAKO) was added as the secondary antibody in 50 μl/wellportions and allowed to react at room temperature for 1 hour. After thereaction and subsequent washing with Tween-PBS, the ABTS substratesolution [a solution prepared by dissolving 0.55 g of2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt in 1liter of 0.1 M citrate buffer (pH 4.2), and further adding thereto 1μl/ml of hydrogen peroxide just before use] was added in 50 μl/wellportions to effect color development, and then OD415 was measured usinga plate reader Emax (manufactured by Molecular Devices). The results areexpressed by relative values (%) wherein OD415 when an antibody alone isadded is defined as 100.

As shown in FIG. 3, binding of the anti-hIGF antibody KM1468 to hIGF-Iwas inhibited by hIGF-I in a concentration-dependent manner, but theinhibitory activity was not observed in these peptides regardless ofusing alone or combination. The above results strongly suggest thatKM1468 does not merely recognaize an amino acid primary sequence ofhIGF-I but recognizes three-dimensional structure of hIGF-I.

(3) Verification of Cross Reactivity of Anti-hIGF Antibody KM1468 byCompetitive ELISA

Cross reactivity of the purified anti-hIGF antibody KM1468 with hIGF-IIand human insulin was examined by the competitive ELISA shown below. Asthe antigens, hIGF-I (manufactured by Pepro Tech), hIGF-II (manufacturedby Pepro Tech) and human insulin (manufactured Wako Pure ChemicalIndustries) were used.

The methylated BSA-hIGF-I antigen prepared in Example 1(1) or amethylated BSA-hIGF-II antigen prepared in the same manner as in Example1(1) was immobilized on a plate in accordance with the method shown inExample 1(4), at a concentration of 0.1 μg/ml in the case of themethylated BSA-hIGF-I antigen, or at a concentration of 1.0 μg/ml in thecase of the methylated BSA-hIGF-II antigen, KM1468 diluted to 0.6 μg/mlwas dispensed therein in 25 μl/well portions, and then each of 4-foldserial dilutions of hIGF-I, hIGF-II or human insulin prepared bystarting from 20 μg/ml was dispensed in 25 μl/well portions, mixed andallowed to react at room temperature for 1 hour. After the reaction andsubsequent washing with Tween-PBS, 1,000 times-dilutedperoxidase-labeled rabbit anti-rat Ig antibody (manufactured by DAKO)was added in 50 μl/well portions as the secondary antibody in the caseof the anti-hIGF antibody KM1468. After the reaction and subsequentwashing with Tween-PBS, the ABTS substrate solution [a solution preparedby dissolving 0.55 g of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonicacid) ammonium salt in 1 liter of 0.1 M citrate buffer (pH 4.2), andfurther adding thereto 1 μl/ml of hydrogen peroxide just before use] wasadded in 50 μl/well portions to effect color development, and then OD415was measured using a plate reader Emax (manufactured by MolecularDevices). The results are represented by relative values (%) whereinOD415 when an antibody alone is added is defined as 100.

The results are shown in FIG. 4. As shown in FIG. 4A, binding of theanti-hIGF antibody KM1468 to hIGF-I was strongly inhibited by hIGF-I andhIGF-II. In the same manner, as shown in FIG. 4B, binding of theanti-hIGF antibody KM1468 to hIGF-II was strongly inhibited by hIGF-Iand hIGF-II. In addition, these inhibitions by hIGF-I and hIGF-II werethe same degree. That is, it is shown that the anti-hIGF antibody KM1468can react with both of hIGF-I and hIGF-II by almost the same strength.On the other hand, binding of the anti-hIGF antibody KM1468 to hIGF-I orhIGF-II was not inhibited by human insulin.

Example 3

Verification of Reactivity of Anti-hIGF Antibody with IGF

Comparison of the reactivity of KM1468 and two commercially availableanti-hIGF antibodies with antigens was carried out in the followingmanner. As the antibodies, the anti-hIGF antibody KM1468, sm1.2 as acommercially available anti-hIGF-I antibody (manufactured by UpstateBiotechnology) and S1F2 as a commercially available anti-hIGF-IIantibody (manufactured by Upstate Biotechnology) were used. As theantigens, hIGF-I (manufactured by Pepro Tech), hIGF-II (manufactured byPepro Tech) and human insulin (manufactured by Wako Pure ChemicalIndustries) were used.

(1) Measurement of Binding Strength Using Surface Plasmon Resonance

In order to analyze binding activity of the anti-hIGF antibody KM1468 toan antigen hIGF-I or hIGF-II, binding strengths of the anti-hIGFantibody KM1468, a commercially available anti-hIGF-I antibody sm1.2 anda commercially available anti-hIGF-II antibody S1F2 to hIGF-I andhIGF-II were measured in the following manner using the biosensorBiacore 2000 (manufactured by BIACORE) use of a surface plasmonresonance. HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20,pH 7.4) (manufactured by BIACORE) was used for the dilution of analytesand as the reaction buffer.

Using an amine coupling (manufactured by BIACORE) in a sensor tip CM-5(manufactured by BIACORE), hIGF-I was immobilized in 36.0 pg/mm², orhIGF-II was immobilized in 41.7 pg/mm², three antibodies diluted 6 stepsby 2-fold dilution starting from 20 μg/ml were added thereto as theanalytes at a flow rate of 20 μl/minute for 2 minutes, and thendissociation of the analytes was observed for 5 minutes. The reactionwas carried out at 25° C. Association rate constant Kass anddissociation constant Kdiss were calculated from the binding reactioncurves at respective concentrations, and the binding constant K_(A)(M⁻¹) of each of these antibodies was calculated. The binding constantK_(A) is calculated by K_(A)=Kass/Kdiss.

TABLE 1 KM1468 sm1.2 S1F2 K_(A) (hIGF-I) 7.86 × 10⁹ 1.86 × 10⁸ 4.62 ×10⁸ K_(A) (hIGF-II) 8.63 × 10⁹ 7.35 × 10⁷ 2.40 × 10⁹

The results are shown in Table 1. The K_(A) value of the anti-hIGFantibody KM1468 to hIGF-I was 7.86×10⁹ M⁻¹, and its K_(A) value tohIGF-II was 8.63×10⁹ M⁻¹. Since the K_(A) ratio of KM1468 to hIGF-I andhIGF-II was almost 1:1, it was shown that KM1468 can bind strongly toboth of hIGF-I and hIGF-II with an almost equivalent strength. On theother hand, the K_(A) value of the commercially available anti-hIGF-Imonoclonal antibody sm1.2 to hIGF-I was 1.86×10⁸ M⁻¹, and its K_(A)value to hIGF-II was 7.35×10⁷ M⁻¹. The K_(A) values of the anti-hIGFantibody KM1468 to hIGF-I and hIGF-II were about 42 times higher tohIGF-I and about 120 times higher to hIGF-II, in comparison with theK_(A) value of the commercially available anti-hIGF-I antibody sm1.2.Also, the K_(A) value of the commercially available anti-hIGF-IIantibody S1F2 to hIGF-I was 4.62×10⁸ M⁻¹, and its K_(A) value to hIGF-IIwas 2.4×10⁹ M⁻¹. The K_(A) values of the anti-hIGF antibody KM1468 tohIGF-I and hIGF-II were about 18 times higher to hIGF-I and about 3.6times higher to hIGF-II, in comparison with the K_(A) value of thecommercially available anti-hIGF-II antibody S1F2. That is, it was shownthat the anti-hIGF antibody KM1468 has a strong binding activity to eachof hIGF-I and hIGF-II, in comparison with the commercially availableanti-hIGF-I antibody sm1.2 and the commercially available anti-hIGF-IIantibody S1F2.

(2) Influence of Anti-hIGF Antibody upon hIGF-dependent Growth

Each of a human breast cancer cell line MCF7 (ATCC HTB-22), a humancolon cancer cell line HT-29 (ATCC HTB-38) or a human osteosarcoma cellline MG-63 (ATCC CRL-1427) was prepared into a TF/BSA medium [a mediumprepared by adding 10 μg/ml of human transferrin (manufactured by GibcoBRL) and 200 μg/ml of BSA to D-MEM/F-12 (manufactured by Gibco BRL)] ata density of 0.5 to 1×10⁵ cells/ml and dispensed in 100 μl/well portionsinto a 96 well culture plate. Subsequently, each of the factors hIGF-I(manufactured by Pepro Tech), hIGF-II (manufactured by Pepro Tech) andhuman insulin (manufactured Wako Pure Chemical Industries) diluted toeach concentration with the TF/BSA medium was added in 50 μl/wellportions thereto, and each of the antibodies diluted to eachconcentration with the TF/BSA medium in 50 μl/well portions, andcultured at 37° C. for 5 days in a 5% CO₂ incubator. After theculturing, a cell proliferation reagent WST-1 (manufactured by Roche)was dispensed in 20 μl/well portions, the cells were further cultured at37° C. for 2.5 to 4 hours in a 5% CO₂ incubator, and then the absorbanceat OD450 nm (hereinafter referred to as OD450) was measured using aplate reader Emax (manufactured by Molecular Devices).

Growth curves of the human breast cancer cell line MCF7 by respectivefactors are shown in FIG. 5A. In addition, growths in the presence ofrespective antibodies are shown in FIG. 5B in the presence of 40 ng/mlof hIGF-I, in FIG. 5C in the presence of 100 ng/ml of hIGF-II and inFIG. 5D in the presence of 100 ng/ml of human insulin. As shown in FIG.5, the anti-hIGF antibody KM1468 strongly inhibited the cell growth byhIGF-I and hIGF-II, and the growth inhibitory activity was higher thanthe commercially available anti-hIGF-I antibody sm1.2 and thecommercially available anti-hIGF-II antibody S1F2. On the other hand,each of the antibodies did not exert influence upon the growth by humaninsulin. The above results correlated well with the bindingspecificities of respective antibodies observed by the competitive ELISAof Example 3(1) and (2), and distinctly showed that the functions ofhIGF-I and hIGF-II are inhibited by the binding of each antibody.

Growth curves of the human colon cancer cell line HT-29 by respectivefactors are shown in FIG. 6A. In addition, growths in the presence ofrespective antibodies are shown in FIG. 6B in the presence of 10 ng/mlof hIGF-I, in FIG. 6C in the presence of 10 ng/ml of hIGF-II and in FIG.6D in the presence of 20 ng/ml of human insulin.

As shown in FIG. 6, the anti-hIGF antibody KM1468 strongly inhibited thecell growth by hIGF-I and hIGF-II to almost the same degree, and thegrowth inhibitory activity was higher than the commercially availableanti-hIGF-I antibody sm1.2 and the commercially available anti-hIGF-IIantibody S1F2. On the other hand, each of the antibodies did not exertinfluence upon the growth by human insulin. The above results correlatedwell with the binding specificities of respective antibodies observed bythe competitive ELISA of Example 3(1) and (2), and distinctly showedthat the functions of hIGF-I and hIGF-II are inhibited by the binding ofeach antibody. In addition, when KM1468 was added to the culturing ofHT-29 cell in the presence of the addition of hIGF-I shown in FIG. 6B,and when KM1468 or S1F2 was added to the culturing of HT-29 cell in thepresence of hIGF-II shown in FIG. 6C, growth of the cells was inhibitedin comparison with the cell growth in the absence of respectiveantibodies and respective growth factors shown by dotted lines. That is,the HT-29 cell grows by producing hIGF-I or hIGF-II by itself, and theanti-hIGF antibody can inhibit an effect to proliferate cells by agrowth factor produced by a cell itself.

Growth curves of the human osteosarcoma cell strain MG-63 by respectivefactors are shown in FIG. 7A. In addition, growths in the presence ofthe addition of respective antibodies are shown in FIG. 7B in thepresence of 20 ng/ml of hIGF-I, in FIG. 7C in the presence of 20 ng/mlof hIGF-II and in FIG. 7D in the presence of 20 ng/ml of human insulin.As shown in FIG. 7, the anti-hIGF antibody KM1468 strongly inhibited thecell growth by hIGF-I and hIGF-II to almost the same degree, and thegrowth inhibitory activity was higher than the commercially availableanti-hIGF-I antibody sm1.2 and the commercially available anti-hIGF-IIantibody S1F2. On the other hand, each of the antibodies did not exertinfluence upon the growth by human insulin. The above results correlatedwell with the binding specificities of respective antibodies observed bythe competitive ELISA of Example 3(1) and (2), and distinctly showedthat the functions of hIGF-I and hIGF-II are inhibited by the binding ofeach antibody.

The hIGF-I- or hIGF-II-dependent cell growth inhibitory activity in thecase of the above three types of cells was observed in any one of theanti-hIGF antibody KM1468, the commercially available anti-hIGF-Iantibody sm1.2 and the commercially available anti-hIGF-II antibodyS1F2. In the case of the hIGF-1-dependent growth activity, this cellgrowth inhibitory activity was highest in the anti-hIGF antibody KM1468,followed by the anti-hIGF-I antibody sm1.2 and the anti-hIGF-II antibodyS1F2. Also, in the case of the hIGF-II-dependent growth activity, thiscell growth inhibitory activity was highest in the anti-hIGF antibodyKM1468, followed by the anti-hIGF-II antibody S1F2 and the anti-hIGF-Iantibody sm1.2. This result coincides well with the result of bindingstrengths obtained using the surface plasmon resonance in Example 3(1),and distinctly showed that the anti-hIGF antibody KM1468 is superior tothe commercially available antibodies of its binding activity to both ofhIGF-I and hIGF-II and its hIGF-1-dependent or hIGF-II-dependent cellgrowth inhibitory effect.

Example 4

Influence of Anti-hIGF Antibody KM1468 upon Growth of hIGF-I ExpressingCell

(1) Construction of hIGF-I Expressing Cell

A transformant in which hIGF-I gene was transferred into a human lungcancer cell line A549 (ATCC CCL-185) was prepared in the followingmanner.

(1-1) Cloning of hIGF-I Gene and Preparation of Expression Vector

A 45.6 μg portion of total RNA was prepared from 1×10⁷ cells of a humanlung cancer cell strain PC-9 (British Journal of Cancer, 39, 15, 1976)using an RNA preparation kit RNeasy (manufactured by QIAGEN) inaccordance with the instructions attached thereto. Using a 5 μg portionof the prepared total RNA, cDNA was synthesized using Superscript II(manufactured by GIBCO-BRL) in accordance with the instructions attachedthereto.

Using the synthesized cDNA as the template, the hIGF-I gene was clonedby PCR. As primers for hIGF-I gene amplification, synthetic DNAsrespectively having the nucleotide sequences shown in SEQ ID NOS: 27 and28 were designed. Each synthetic DNAs contains a restriction enzymerecognizing sequence in its 5′-terminal for cloning it into plasmidspBluescript II SK(−) (manufactured by Stratagene) and pKANTEX93 (WO97/10354). Specifically, 20 ng of the cDNA synthesized from the humanlung cancer cell line PC-9, obtained in the above, was added to a buffersolution containing 50 μl of KOD(+) DNA Polymerase-attached KOD(+)Buffer #1 (manufactured by TOYOBO), 0.2 mM dNTPs, 2 mM magnesiumchloride and 1 μM of the synthetic DNA respectively having thenucleotide sequences shown in SEQ ID NOS: 27 and 28, and using a DNAthermal cycler GeneAmp PCR System 9600 (manufactured by PERKIN ELMER),the mixture was heated at 94° C. for 1 minute, and then, by adding 2.5units of KOD DNA Polymerase (manufactured by TOYOBO), a cycle of 30seconds at 94° C., 30 seconds at 62° C. and 30 seconds at 72° C. wasrepeated 30 cycles. A 50 μl portion of each reaction solution wasdigested with restriction enzymes EcoRI (manufactured by Takara Shuzo)and SalI (manufactured by Takara Shuzo) and then subjected to an agarosegel electrophoresis, and a PCR product of a gene coding for hIGF-I ofabout 0.5 kb was recovered using QIAquick Gel Extraction Kit(manufactured By QIAGEN).

Next, 0.1 μg of DNA obtained by digesting the plasmid pBluescript IISK(−) (manufactured by Stratagene) with the restriction enzymes EcoRIand SalI and then dephosphorylating the termini with Calf IntestineAlkaline Phosphatase (manufactured By Takara Shuzo, hereinafter referredto as CIAP) and 0.1 μg of each PCR product obtained in the above wereprepared into 7.5 μl by adding sterile water and then allowed to reactat 16° C. overnight by adding 7.5 μl of Ligation High (manufactured byTOYOBO). Using the recombinant plasmid DNA solution obtained in thismanner, an Escherichia coli DH5α strain (manufactured by TOYOBO) wastransformed. Each plasmid DNA was prepared from the transformant, whichsubjected to the reaction using BigDye Terminator Cycle Sequencing FSReady Reaction Kit (manufactured by Applied Biosystems) in accordancewith the instructions attached thereto, and its nucleotide sequence wasdetermined using a nucleotide sequence automatic analyzer ABI PRISM 377(manufactured by Applied Biosystems). As a result, the plasmid ofinterest pBS(II)SK(−)/hIGF-I having a gene sequence coding for hIGF-Ishown in FIG. 8 was obtained.

Next, the restriction enzyme fragment (EcoRI-KpnI) of the pBS(II)SK(−)/hIGF-I obtained in the above coding for hIGF-I ligated with theEcoRI-KpnI fragment of pKANTEX93, and a plasmid pKANTEX93/hIGF-I shownin FIG. 8 was constructed. Nucleotide sequence of the plasmidpKANTEX93/hIGF-I was determined in the same manner as described aboveusing the nucleotide sequence automatic analyzer ABI PRISM 377. As aresult, the plasmid of interest pKANTEX93/hIGF-I containing a genecoding for hIGF-I was obtained.

(1-2) Preparation of hIGF-I Transformant

An hIGF-I expressing cell was prepared in the following manner byintroducing the plasmid pKANTEX93/hIGF-I obtained in Example 1(1-1) intoan animal cell.

The plasmid pKANTEX93/hIGF-I was digested with a restriction enzymeAatII (manufactured by TOYOBO) to linearize, and an 8 μg portion thereofwas introduced into 4×10⁶ cells of the human lung cancer cell line A 549(ATCC CCL-185) by the electroporation method (Cytotechnology, 3,133-140, 1990), and then the cells were suspended in 15 ml of RPMImedium [RPMI 1640 medium (manufactured by Invitrogen) containing 10% FCSand 50 μg/ml gentamicin (manufactured by Nakalai Tesque)] andtransferred into a T75 flask (manufactured by Sumilon). After 24 hoursof culturing at 37° C. in a 5% CO₂ incubator, G418 was added thereto toa concentration of 0.2 mg/ml and further cultured for 1 to 2 weeks. AnA549/hIGF-I transformant having G418 resistance (hereinafter referred toas A549/hIGF-I) was obtained.

(1-3) Determination of hIGF-I Produced in a Culture Supernatant ofA549/hIGF-I Cell

The following test was carried out in order to verify whether theintroduced hIGF-I gene is expressed in the A549/hIGF-I cell prepared inExample 3(1-1) and said cell is producing hIGF-I.

The A549/hIGF-I cell or A549 cell was cultured in the RPMI medium, andthen the culture supernatant was recovered to measure the amount ofhIGF-I contained in the culture supernatant by ELISA method as follows.

The methylated BSA-hIGF-I-immobilized plate shown in Example 1(4) wasprepared, an hIGF-I solution prepared by 5-fold serial dilution startingfrom 2 μg/ml as the positive sample, or a culture supernatant ofA549/hIGF-I or A549 cell, was dispensed in 25 μl/well portions, and thenpurified antibody of the anti-hIGF antibody KM1468 diluted to 0.6 μg/mlwas dispensed, mixed and allowed to react at room temperature for 2hours. After the reaction and subsequent washing with Tween-PBS, 1,000times-diluted peroxidase-labeled rabbit anti-rat Ig antibody(manufactured by DAKO) was dispensed in 50 μl/well portions and allowedto react at room temperature for 1 hour. After the reaction andsubsequent washing with Tween-PBS, 1,000 times-diluted anti-rat IgG-HRP(manufactured by DAKO) was dispensed in 50 μl/well portions and allowedto react at room temperature for 1 hour. After the reaction andsubsequent 5 times of washing with Tween-PBS, an ABTS substrate solution[a solution prepared by dissolving 0.55 g of2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt in 1liter of 0.1 M citrate buffer (pH 4.2), and further adding thereto 1μl/ml of hydrogen peroxide just before use] was added in 50 μl/wellportions to effect color development, and then OD415 was measured usingthe plate reader Emax.

The results are shown in FIG. 9. As shown in FIG. 9A, in comparison withthe culture supernatant of A549 cell to which with the hIGF-I gene wasnot introduced, the binding activity was distinctively reduced in theculture supernatant of A549/hIGF-I cell to which the hIGF-I gene wasintroduced, thus showing that the A549/hIGF-I cell expresses hIGF-I.

(1-4) Influence of Anti-hIGF Antibody KM1468 upon Growth of hIGF-IExpressing Cell

Whether the KM1468 can inhibit a cell growth dependent on hIGF-Iproduced by the cell itself (hereinafter referred to as autocrine cellgrowth) was examined using the hIGF-I gene-introduced cell A549/hIGF-Icell prepared in Example 3(1-1).

The A549/hIGF-I cell or A549 cell was cultured using RPMI 1640 medium(manufactured by Invitrogen) containing 10% FCS and 50 μg/ml gentamicin(manufactured by Nakalai Tesque) (hereinafter referred to as RPMImedium), and then respectively suspended in DMEM/F12 medium (-FCS,-Phenol red) (manufactured by Invitrogen) containing 10 μg/ml humantransferrin (manufactured by GIBCO) and 200 μg/ml BSA (manufactured byInvitrogen) (to be referred to as serum-free medium hereinafter) to acell density of 2×10⁵ cells/ml.

Cell suspension of the A549/hIGF-I cell or A549 cell was dispensed in100 μl/well portions into a 96 well plate (manufactured by Sumilon), theanti-hIGF antibody KM1468 serially diluted with the serum-free medium by5-fold dilution starting from 200 μg/ml was added in 100 μl/wellportions to each well, and then the cells were cultured at 37° C. for 5days in a 5% CO₂ incubator. After the culturing, a cell proliferationreagent WST-1 (manufactured by Roche) was dispensed in 200 μl/wellportions, the cells were further cultured at 37° C. for 4 hours in the5% CO₂ incubator, and then the absorbance at OD450 nm (hereinafterreferred to as OD450) was measured using a plate reader Emax(manufactured by Molecular Devices).

The results are shown in FIG. 10. The abscissa shows concentration ofthe anti-hIGF antibody KM1468 in each well at the time of the culturing.Growth of the A549/hIGF-I cell in the absence of the anti-hIGF antibodyKM1468 shown by broken line was evidently increased in comparison withthe growth of A549 cell shown by solid line which does not producehIGF-I. This shows an autocrine growth in which the A549/hIGF-I cellprompts growth of the A549/hIGF-I cell itself by the self-producedhIGF-I. Such an autocrine growth shown in FIG. 10 was dose-dependentlyinhibited when the antibody KM1468 was added at the time of theculturing of A549/hIGF-I cell. On the other hand, the antibody KM1468did not exert influence upon the growth of A549 cell. That is, it wasshown that the anti-hIGF antibody KM1468 can inhibit the autocrine cellgrowth by the hIGF-I produced by the cell itself.

(1-5) Influence of Anti-hIGF Antibody KM1468 upon Anchorage IndependentGrowth of hIGF-I Expressing Cell

Cells after malignant alteration have the ability to perform anchorageindependent growth in which they can grow regardless of a suspendedcondition with no cell engraftment, such as in a soft agar. The abilityto perform anchorage independent growth is very closely related to thetumorigenicity of cells, and it is considered that hIGF-I is concernedtherein. Whether the KM1468 can inhibit anchorage independent growth ofa cell was examined in the following manner using the A549/hIGF-I cellprepared in Example 3(1-1).

RPMI medium containing warmed 0.3% agar noble (manufactured by Difco)(hereinafter referred to as agar-RPMI medium) was dispensed in 1 ml/wellportions into a 12 well plate (manufactured by Costar), and allowing themedium to stand at room temperature for scores of minutes to effectgelation. After culturing the A549/hIGF-I cell or A549 cell using theRPMI medium, the resulting cells were suspended in warmed agar-RPMImedium to a cell density of 1×10³ cells/ml.

The cell suspension of A549/hIGF-I cell or A549 cell was overlaid oneach well in an amount of 1 ml/well. After allowing to stand at roomtemperature for several minutes to effect gelation, the cells werecultured at 37° C. for 4 weeks in a 5% CO₂ incubator. After theculturing, the number of colonies formed in each well was counted undera microscope.

The results are shown in FIG. 11. As shown in FIG. 11, the anchorageindependent cell growth of the A549/hIGF-I cell producing hIGF-1 wasincreased in comparison with the anchorage independent cell growth ofthe A549 cell. In addition, when 10 μg/ml of the anti-hIGF antibodyKM1468 was added during culturing of A549/hIGF-I cell in the soft agar,the anchorage independent cell growth was completely inhibited by theaddition of KM1468. That is, it was shown that hIGF-I is concerned inthe anchorage independent cell growth, and the hIGF-dependent anchorageindependent cell growth is inhibited by the anti-hIGF antibody KM1468.

(1-6) Tumor Growth Inhibitory Effect of Anti-hIGF Antibody KM1468 uponhIGF-I Expressing Cell

Using the A549/hIGF-I cell prepared in Example 3(1-1), tumor growthinhibitory effect of the anti-hIGF antibody KM1468 was examined in thein vivo tumor formation in which hIGF-I takes a role according to thefollowing manner.

The A549/hIGF-I cell or A549 cell was cultured using the RPMI medium andthen respectively suspended in PBS to a cell density of 1×10⁶ cells/ml.

A 100 μl portion of the cell suspension of A549/hIGF-I cell or A549 cellwas subcutaneously transplanted into the right thoracic region of eachnude mouse Balb/c Ajc-1 nu (female) of 6-weeks-old. The number oftransplanted cells per one mouse becomes 1×10⁷ cells. Starting justafter the transplantation, 500 μg per one mouse of the anti-hIGFantibody KM1468 was administered through the tail vein twice a week, 8times in total. As a negative control, PBS was simultaneouslyadministered to on the same subcutaneous tumor transplantation mouse.Five days after the cell transplantation, tumor volume was measured. Thetumor volume (mm³) was calculated from the length, breadth and height ofthe tumor using a formula of length×breadth×height×0.5236.

The results are shown in FIG. 12. When growth of the subcutaneous tumorin the mouse transplanted with the A549 cell which does not producehIGF-I was compared with that of the mouse transplanted with theA549/hIGF-I cell which produces hIGF-I, growth of the tumor wasincreased in the case of the subcutaneous tumor in the mousetransplanted with the A549/hIGF-I cell. In addition, in the mousetransplanted with the A549/hIGF-I cell, growth of the subcutaneous tumorwas significantly inhibited when the anti-hIGF antibody KM1468 wasadministered. This result distinctively shows that the anti-hIGFantibody KM1468 inhibits growth of tumor also in vivo due to inhibitionof hIGF-I.

Example 5

Preparation of Anti-hIGF-I Chimeric Antibody

(1) Isolation and Analysis of cDNA Coding for the V Region of Anti-hIGFAntibody KM1468

(1-1) Preparation of mRNA from Anti-hIGF Antibody KM1468 ProducingHybridoma

A 27 μg portion of KM1468-derived mRNA was prepared from 5×10⁷ cells ofan anti-hIGF antibody KM1468 producing hybridoma KM1468 (FERM BP 7978)using an mRNA preparation kit Fast Track mRNA Isolation Kit(manufactured by Invitrogen) in accordance with the instructionsattached thereto.

(1-2) Preparation of H Chain and L Chain cDNA Libraries of Anti-hIGFAntibody KM1468

A cDNA having an EcoRI-NotI adapter sequence on both termini wassynthesized from 5 μg of the KM1468 mRNA prepared in Example 5(1-1)using TimeSaver cDNA Synthesis Kit (manufactured by Amersham Pharmacia)in accordance with the instructions attached thereto. Total amount ofthe synthesized cDNA was dissolved in 20 μl of sterile water and thenfractionated by an agarose gel electrophoresis, and a cDNA fragment ofabout 1.5 kb corresponding to the H chain of an IgG class antibody andcDNA fragment of about 1.0 kb corresponding to the L chain of a κ classwere recovered in an amount of about 1.0 μl respectively using QIAquickGel Extraction Kit (manufactured by QIAGEN). Next, using λZAPIIPredigested EcoRI/CIAP-Treated Vector Kit (manufactured by Stratagene),each of 0.1 μg of the cDNA fragment of about 1.5 kb and 0.1 μg of thecDNA fragment of about 1.0 kb was ligated to 1 μg of λZAPII vector whosetermini had been dephosphorylated with Calf Intestine AlkalinePhosphatase after digestion with a restriction enzyme EcoRI attached tothe kit, in accordance with the instructions attached thereto. After theligation, a 2.5 μl of each reaction solution was packaged into λ phageusing Gigapack III Gold Packaging Extracts (manufactured by Stratagene)in accordance with the instructions attached thereto to therebyobtaining 5.0×10⁴ phage clones as an H chain cDNA library of KM1468, and4.0×10⁴ phage clones as an L chain cDNA library. Next, each phage wasimmobilized on a nylon membrane filter Hybond-N⁺ (manufactured byAmersham Pharmacia) in accordance with a conventional method (MolecularCloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York,1989).

(1-3) Cloning of H Chain and L Chain cDNA of Anti-hIGF Antibody KM1468

The nylon membrane filters of H chain cDNA library and L chain cDNAlibrary of KM1468 prepared in Example 5 (1-2) were detected using a cDNAof the C region of a mouse antibody [H chain is a fragment of mouse Cγ2bcDNA (Nature, 283, 786-789, 1980), and L chain is a fragment of mouse CκcDNA (Cell, 22, 197-207, 1980)] as the probe using ECL Direct NucleicAcid Labeling and Detection Systems (manufactured by Amersham Pharmacia)in accordance with the instructions attached thereto, and each 10 phageclones strongly hybridized to the probe were obtained for each of the Hchain and L chain. Next, each phage clone was converted into plasmid bythe in vivo excision method in accordance with the instructions ofλZAPII Predigested EcoRI/CIAP-Treated Vector Kit (manufactured byStratagene). Nucleotide sequence of cDNA contained in the obtainedplasmid was determined by carrying out the reaction using BigDyeTerminator Cycle Sequencing FS Ready Reaction Kit (manufactured byApplied Biosystems) in accordance with the instructions attachedthereto, and using a nucleotide sequence automatic analyzer ABI PRISM377 (manufactured by Applied Biosystems). As a result, a plasmidpKM1468H5-2 containing the full length of functional H chain cDNA and aplasmid pKM1468L5-1 containing the full length of functional L chaincDNA, in which an ATG sequence considered to be the initiation codon ispresent in the 5′-terminus of respective cDNA, were obtained.

(1-4) Analysis of V Region Amino Acid Sequences of Anti-hIGF AntibodyKM1468

The full length nucleotide sequence of VH of KM1468 contained in theplasmid pKM1468H5-2 is shown in SEQ ID NO: 1 and full length amino acidsequence of VH of KM1468 deduced therefrom is shown in SEQ ID NO: 2, andfull length nucleotide sequence of VL of KM1468 contained in the plasmidpKM1468L5-1 is shown in SEQ ID NO: 3 and full length amino acid sequenceof VL of KM1468 deduced therefrom is shown in SEQ ID NO: 4,respectively. In this connection, there are a large number of nucleotidesequences respectively corresponding to the amino acid sequences shownby SEQ ID NOS: 2 and 4, other than those shown by SEQ ID NOS: 1 and 3,and all of them are included in the scope of the present invention.Based on the comparison with known sequence data of antibodies(Sequences of Proteins of Immunological Interest, US Dept. Health andHuman Services, 1991) and or the comparison with results of the analysisof N-terminal amino acid sequences of VH and VL of the purifiedanti-hIGF antibody KM1468 using a protein sequencer PPSQ-10(manufactured by Shimadzu), it was revealed that the isolated respectivecDNA is a secretion signal sequence-containing full length cDNA codingfor the H chain or L chain of the anti-hIGF antibody KM1468, and asequence of from the -19th to -1st positions of the amino acid sequenceshown by SEQ ID NO: 2 is the secretion signal sequence of VH, and asequence of from the -22nd to -1st positions of the amino acid sequenceshown by SEQ ID NO: 4 is the secration signal sequense of VL.

Next, novelty of the VH and VL amino acid sequences of the anti-hIGFantibody KM1468 was examined. Using GCG Package (version 10.0,manufactured by Genetics Computer Group) as a sequence analyzing system,the existing protein amino acid sequence data bases [SWISS-PROT (Release39.0), PIR-Protein (Release 65.0)] were searched by the BLAST method(Journal of Molecular Biology, 215,403-410, 1990). As a result,completely coincided sequences were not found for both VH and VL, and itwas confirmed that the VH and VL of the anti-hIGF antibody KM1468 arenovel amino acid sequences.

In addition, CDRs of the VH and VL of the anti-hIGF antibody KM1468 wereidentified by comparing with amino acid sequences of known antibodies.Amino acid sequences of CDR1, 2 and 3 of the VH of KM1468 are shown inSEQ ID NOS: 5, 6 and 7, and amino acid sequences of CDR1, 2 and 3 of theVL in SEQ ID NOS: 8, 9 and 10, respectively.

(2) Construction of Human Chimeric Antibody Expression Vector

An anti-hIGF-I chimeric antibody expression vector derived from theanti-hIGF antibody KM1468 was constructed in the following manner usingthe vector for expression of humanized antibody pKANTEX93 described inWO 97/10354 which can express the human IgG1, κ class antibodies and theplasmids obtained in Example 5(1-3) containing cDNAs for the H chain andL chain of KM1468.

Firstly, in order to insert the cDNAs for the VH and VL of KM1468 intothe expression vector pKANTEX93 such that the amino acid sequences arenot changed, cDNAs for the VH and VL of KM1468 were reconstructed byPCR. As the primers, synthetic DNAs respectively having the nucleotidesequences of SEQ ID NOS: 11 and 12 were designed for the VH cDNA, andsynthetic DNAs respectively having the nucleotide sequences of SEQ IDNOS: 13 and 14 were designed for the VL cDNA. Each of the synthetic DNAscontains a restriction enzyme recognizing sequence in the 5′-terminusfor its cloning into pKANTEX93. Specifically, 20 ng of the plasmidpKM1468H5-2 obtained in Example 5(1-3) was added to a buffer solutioncontaining 50 μl of KOD DNA Polymerase-attached PCR Buffer #1(manufactured by TOYOBO), 0.2 mM dNTPs, 1 mM magnesium chloride and 0.5μM of the synthetic DNAs having the nucleotide sequences shown in SEQ IDNOS: 11 and 12, and using a DNA thermal cycler GeneAmp PCR System 9600(manufactured by PERKIN ELMER), the mixture was heated at 94° C. for 3minutes, to which 2.5 units of KOD DNA Polymerase (manufactured byTOYOBO) was added, and a cycle of 15 seconds at 98° C., 2 seconds at 65°C. and 30 seconds at 74° C. was repeated 25 cycles. In the same manner,another PCR was carried out by the same method described in the above,by adding 20 ng of the plasmid pKM1468L5-1 obtained in Example 5(1-3) toa buffer solution containing 50 μl of KOD DNA Polymerase-attached PCRBuffer #1 (manufactured by TOYOBO), 0.2 mM dNTPs, 1 mM magnesiumchloride and 0.5 μM of the synthetic DNA fragments having the nucleotidesequences shown in SEQ ID NOS: 13 and 14. A 10 μl portion of eachreaction solution was subjected to an agarose gel electrophoresis, andthen a PCR product of about 0.5 kb for VH or a PCR product of about 0.43kb for VL was recovered using QIAquick Gel Extraction Kit (manufacturedBy QIAGEN).

Next, 0.1 μl of DNA obtained by digesting the plasmid pBluescript IISK(−) (manufactured by Stratagene) with the restriction enzyme SmaI(manufactured by Takara Shuzo) and then dephosphorylating the terminiwith Calf Intestine Alkaline Phosphatase (hereinafter referred to asCIAP hereinafter; manufactured by Takara Shuzo) and 0.1 μl of each PCRproduct obtained in the above were prepared into 7.5 μl by addingsterile water and then allowed to react at 22° C. overnight after adding7.5 μl of the solution I of TaKaRa DNA Ligation Kit Ver. 2 (manufacturedby Takara Shuzo) and 0.3 μl of the restriction enzyme SmaI (manufacturedby Takara Shuzo). Using the recombinant plasmid DNA solution obtained inthis manner, an Escherichia coli DH5α strain (manufactured by TOYOBO)was transformed. Each plasmid DNA was prepared from the transformant,its nucleotide sequence was determined by carrying out the reactionusing BigDye Terminator Cycle Sequencing FS Ready Reaction Kit(manufactured by Applied Biosystems) in accordance with the instructionsattached thereto and using a nucleotide sequence automatic analyzer ABIPRISM377 (manufactured by Applied Biosystems). In this manner, plasmidspKM1468VH and pKM1468VL having the nucleotide sequences of interestshown in FIG. 13 were obtained.

Next, a plasmid pKANTEX1468H shown in FIG. 14 was constructed byinserting the restriction enzyme fragment (NotI-ApaI) containing the VHcDNA of pKM1468VH obtained in the above into the NotI-ApaI site of thevector pKANTEX93 for expression of humanized antibody. Also, a plasmidpKANTEX1468Chi shown in FIG. 14 was constructed by inserting therestriction enzyme fragment (EcoRI-BsiWI) containing the VL cDNA ofpKM1468VL obtained in the above into the EcoRI-BsiWI site of the plasmidpKANTEX1468H. Using the plasmid pKANTEX1468Chi, nucleotide sequences ofthe VH and VL cDNA molecules were determined by carrying out thereaction using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit(manufactured by Applied Biosystems) in accordance with the instructionsattached thereto and using the nucleotide sequence automatic analyzerABI PRISM 377 (manufactured by Applied Biosystems), and it was confirmedas a result that plasmids cloned with the VH and VL cDNAs of interestwere obtained.

(3) Stable Expression of Anti-hIGF Chimeric Antibody Using Animal Cell

Using the anti-hIGF chimeric antibody expression vector pKANTEX1468Chiobtained in Example 5(2-1), expression of the anti-hIGF chimericantibody in an animal cell was carried out in the following manner.

The plasmid pKANTEX1468Chi was digested with a restriction enzyme AatII(manufactured by TOYOBO) to linearize, a 10 μl portion thereof wasintroduced into 4×10⁶ cells of a rat myeloma cell line YB2/0 (ATCC CRL1581) by the electroporation method (Cytotechnology, 3, 133-140, 1990),and then the cells were suspended in 40 ml of H-SFM (5) medium [H-SFMmedium (manufactured by Gibco BRL) containing 5% FCS] and dispensed in200 μl/well portions into a 96 well culture plate (manufactured bySumitomo Bakelite). After 24 hours of culturing at 37° C. in a 5% CO₂incubator, G418 was added thereto to a concentration of 0.5 mg/ml andfurther cultured for 1 to 2 weeks. Culture supernatants were recoveredfrom the wells in which transformant colonies showing G418-resistancewere formed and became confluent, and concentration of the anti-hIGFchimeric antibody in the supernatants was measured by the binding ELISAshown in Example 6(1).

Regarding each of the transformants in wells in which expression of theanti-hIGF chimeric antibody was found in the culture supernatants, inorder to increase antigen expression using of a dhfr gene amplificationsystem, each of the transformants was suspended to a density of 1 to2×10⁵ cells/ml in H-SFM(5) containing 0.5 mg/ml of G418 and 50 nM ofmethotrexate (hereinafter referred to as MTX, manufactured by SIGMA)which is an inhibitor of a dhfr gene product dihydrofolate reductase (tobe referred to as DHFR hereinafter), and the suspension was dispensed in1 ml portions into a 24 well culture plate (manufactured by Greiner). Byculturing at 37° C. for 1 to 2 weeks in a 5% CO₂ incubator,transformants showing a resistance to 50 M MTX were induced. When thetransformants became confluent in wells, concentration of the anti-hIGFchimeric antibody in the culture supernatants was measured by thebinding ELISA shown in Example 6(1). The transformants in wells in whichexpression of the anti-hIGF chimeric antibody was found in the culturesupernatants were then cultured in a medium containing 100 nM MTX by thesame method described in the above, and the transformants obtained inthe same manner were further cultured in a medium containing 200 nM tothereby finally obtain a transformant which can grow in the H-SFM(5)containing 0.5 mg/ml of G418 and 200 nM of MTX and can highly expressthe anti-hIGF chimeric antibody. By subjecting the transformant thusobtained to single cell cloning by limiting dilution method twice, atransformant having the highest expression of the anti-hIGF chimericantibody was obtained. As the transformant producing the anti-hIGFchimeric antibody derived from KM1468, KM3002 can be cited. Thetransformant KM3002 was deposited on Apr. 2, 2002, as FERM BP-7996 inInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (postal code 305-8566; Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan).

(4) Purification of Anti-hIGF Chimeric Antibody from Culture Supernatant

The transformant KM3002 obtained in Example 5(3) which expresses theanti-hIGF chimeric antibody was suspended in the H-SFM containing 0.5mg/ml of G418, 200 nM of MTX and 5% of Daigo's GF21 (manufactured byWako Pure Chemical Industries) to a density of 1 to 2×10⁵ cells/ml, anddispensed in 100 ml portions into 175 cm² flasks (manufactured byGreiner). The cells were cultured at 37° C. for 5 to 7 days in a 5% CO₂incubator, and the culture supernatant was recovered when they becameconfluent. By purifying the anti-hIGF chimeric antibody KM3002 fromabout 1 liter of the culture supernatant using Prosep-A (manufactured byBioprocessing) column in accordance with the instructions attachedthereto, about 10.2 mg of purified protein was obtained. About 4 μg ofthe obtained anti-hIGF chimeric antibody KM3002 was subjected to anelectrophoresis in accordance with a known method (Nature, 227, 680-685,1970) to examine its molecular weight and purification degree. Theresults are shown in FIG. 15. From the purified anti-hIGF chimericantibody KM3002, one band corresponding to a molecular weight of about150 kilodaltons (hereinafter referred to as Kd) was observed undernon-reducing condition, and two bands corresponding to about 50 Kd andabout 25 Kd was obtained under reducing condition. These molecularweights coincided with the reports that the IgG class antibody has amolecular weight of about 150 Kd under non-reducing condition, and isdegraded into the H chain having a molecular weight of about 50 Kd andthe L chain having a molecular weight of about 25 Kd under reducingcondition due to cutting of the intramolecular S—S bond (Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1988;Monoclonal Antibodies: Principles and Practice, Academic Press Limited,1996), thus confirming that the anti-hIGF chimeric antibody KM3002 isexpressed as an antibody molecule having proper structure. In addition,as a result of the analysis of N-terminal amino acid sequences of the Hchain and L chain of the purified anti-hIGF chimeric antibody KM3002using a protein sequencer PPSQ-10 (manufactured by Shimadzu), it wasconfirmed that they coincide with the N-terminal amino acid sequences ofthe H chain and L chain of the anti-hIGF antibody KM1468.

Example 6

Examination of Reactivity of Anti-hIGF Chimeric Antibody KM3002

(1) Reactivity of Anti-hIGF Chimeric Antibody KM3002 to hIGF

Reactivity of the anti-hIGF rat antibody KM1468 and the anti-hIGFchimeric antibody KM3002 purified in Example 5(2-3) with hIGF-I wasexamined by the ELISA shown in Example 1(4). In this case, however,concentration of the methylated BSA-hIGF-I immobilized on the ELISAplate was changed to 0.5 μg/ml, and 4,000 times-dilutedperoxidase-labeled rabbit anti-rat Ig antibody (manufactured by DAKO)was used as the secondary antibody in the case of the rat antibody, and1,000 times-diluted peroxidase-labeled mouse anti-human IgG1 antibody(manufactured by Southern Biotechnology) in the case of the chimericantibody. As shown in FIG. 16, the anti-hIGF chimeric antibody KM3002showed an antibody concentration-dependent binding activity to hIGF-I.In addition, it was suggested that its activity is equivalent to theanti-hIGF rat antibody KM1468, though it is difficult to comparedirectly because of the different secondary antibodies.

(2) Influence of Anti-hIGF Chimeric Antibody KM3002 upon hIGF-DependentCell Growth

Influence of the anti-hIGF rat antibody KM1468, the anti-hIGF chimericantibody KM3002 purified in Example 5(2-3), a commercially availableanti-hIGF-I antibody sm1.2 (manufactured by Upstate Biotechnology) and acommercially available anti-hIGF-II antibody S1F2 (manufactured byUpstate Biotechnology) upon hIGF-dependent cell growth was examined bythe same method of Example 3(4). A colon cancer cell line HT-29 (ATCCHTB-38) was used as the human cancer cell line.

Growth of cells with the addition of respective antibodies are shown inFIG. 17A in the presence of 2 ng/ml of hIGF-I, and in FIG. 17B in thepresence of 10 ng/ml of hIGF-II. As shown in FIG. 17, similar to thecase of KM1468, KM3002 strongly inhibited cell growth by hIGF-I andhIGF-II, and the activity was higher than those of the commerciallyavailable anti-hIGF-I antibody sm1.2 and commercially availableanti-hIGF-II antibody S1F2. In addition, the anti-hIGF antibody KM1468having the same variable region of the chimeric antibody KM3002 showedalmost the same growth inhibitory activity. The above results show thatthe anti-hIGF chimeric antibody KM3002 derived from KM1468 maintainsequivalent antigen binding activity and antigen binding specificity tothose of the original rat antibody KM1468 after the chimerization.

Example 7

Preparation of Anti-hIGF CDR-Grafted Antibody

(1) Construction of cDNAs Coding for VH and VL of Anti-hIGF CDR-GraftedAntibody

(1-1) Design of Amino Acid Sequences of the VH and VL Amino AcidSequences of Anti-hIGF CDR-Grafted Antibody

Firstly, amino acid sequence of the VH of anti-hIGF CDR-grafted antibodywas designed as follows. An FR amino acid sequence of the VH of a humanantibody was selected for grafting the CDR amino acid sequence of the VHof the anti-hIGF antibody KM1468 identified in Example 5(1-4). When ahuman antibody FR having the highest homology with the VH FR of theanti-hIGF antibody KM1468 was searched from an official data base, CAM(Proceedings of the National Academy of Sciences of United States ofAmerica, 77, 3239-3243, 1980) showed the highest homology (81.6%).Accordingly, the VH of anti-hIGF CDR-grafted antibody was designed basedon the FR of CAM. In the FR of CAM, there were four positions where theamino acid sequence is not univocally determined (13th position, 74thposition, 77th position and 90th position), and amino acid residueswhich are not common in the human antibody sequences were recognized inthe 3rd position and 40th position. In order to reduce immunogenicity,these amino acid residues were changed into amino acid residues whichare found in human antibodies with a high frequency (Sequences ofProteins of Immunological Interest, US Dept. Health and Human Services,1991). By grafting the VH CDR amino acid sequence of the anti-hIGFantibody KM1468 to an appropriate position of the designed CAM-derivedFR amino acid sequence, the VH amino acid sequence HV.0 of the anti-hIGFCDR-grafted antibody described in SEQ ID NO: 15 was designed.

Next, amino acid sequence of the VL of anti-hIGF CDR-grafted antibodywas designed as follows. An FR amino acid sequence of the VL of a humanantibody was selected for grafting the CDR amino acid sequence of the VLof the anti-hIGF antibody KM1468 identified in Example 5(1-4). Cabat etal. have classified known various VL regions of human antibodies into 4subgroups based on the homology of their amino acid sequences (HSG I toIV), and reported on consensus sequences for respective subgroups(Sequences of Proteins of Immunological Interest, US Dept. Health andHuman Services, 1991). Since there is a possibility that these consensussequences have low immunogenicity in human, it was decided to design theVL amino acid sequence of anti-hIGF CDR-grafted antibody based on theseconsensus sequences. In order to prepare an anti-hIGF CDR-graftedantibody having a higher activity, among the FR amino acid sequences ofthe consensus sequences of four VL subgroups of human antibodies, an FRamino acid sequence having the highest homology with the FR amino acidsequence of the VL of KM1468 was selected. Results of the homologysearch are shown in Table 2. As shown in Table 2, the FR amino acidsequence of the VL region of KM1468 showed the highest homology with thesubgroup IV.

TABLE 2 HSG I HSG II HSG III HSG IV 66.3% 61.3% 66.3% 67.5%

Based on the above results, the VL amino acid sequence LV.0 of theanti-hIGF CDR-grafted antibody shown in SEQ ID NO: 16 was designed bygrafting the VL CDR amino acid sequence of the anti-hIGF antibody KM1468to an appropriate position of the FR amino acid sequence of theconsensus sequence of subgroup IV of the human antibody VL.

The VH amino acid sequence HV.0 and VL amino acid sequence LV.0 of theanti-hIGF CDR-grafted antibody are sequences in which only the CDR aminoacid sequences of the anti-hIGF antibody KM1468 were grafted to theselected FR amino acid sequences of a human antibody. Generally, in thecase of human CDR-grafted antibodies, their activities are reduced inmany cases when CDR amino acid sequences of an antibody of a non-humananimal is grafted alone. In order to avoid this problem, among FR aminoacid residues different between a human antibody and an antibody of anon-human animal, certain amino acid residues considered to exertinfluence upon the activity are grafted together with the CDR amino acidsequences. Accordingly, an examination was carried out on theidentification of the FR amino acid residues considered to exertinfluence upon the activity. Firstly, a three-dimensional structure ofan antibody V region (HV0LV0) comprising the VH amino acid sequence HV.0and VL amino acid sequence LV.0 of the anti-hIGF CDR-grafted antibodydesigned above was constructed using a computer modeling technique.Three-dimensional structure coordinate system was using a software AbM(manufactured by Oxford Molecular), and the three-dimensional structurewas displayed using a software Pro-Explore (manufactured by OxfordMolecular) or RasMo1 (manufactured by Glaxo), in accordance with therespective instructions attached thereto. Also, a computer model of thethree-dimensional structure of the V region of the anti-hIGF antibodyKM1468 was constructed in the same manner. In addition, athree-dimensional structure model which comprises amino acid sequencesin which amino acid residues in the FR amino acid sequences of VH and VLof HV0LV0, which are different from those in the anti-hIGF antibodyKM1468, were changed in order into the residues positioned in thecorresponding positions of the anti-hIGF antibody KM1468 was constructedin the same manner, and V region three-dimensional structures of theanti-hIGF antibody KM1468, HV0LV0 and modified HV0LV0 were compared. Asa result, as the residues considered to be exerting influence upon theantibody activity by changing three-dimensional structure of the antigenbinding region, among amino acid residues of the FR of HV0LV0, the 1stposition Gln, the 77th position Asn, the 84th position Asn, the 93rdposition Val, the 97th position Ala and the 98th position Arg wereselected regarding the HV.0, and the 1st position Asp, the 9th positionAsp, the 10th position Ser, the 11th position Leu, the 22nd positionAsn, the 35th position Tyr, the 39th position Pro, the 42nd positionPro, the 45th position Leu, the 46th position Leu, the 69th positionAsp, the 70th position Phe, the 71st position Thr, the 82nd position Valand the 84th position Val regarding the LV.0. Among these selected aminoacid residues, at least one or more thereof were changed into the aminoacid residues found in the rat antibody KM1468 to thereby design VH andVL of the human CDR-grafted antibody having various modifications.

(1-2) Construction of cDNA Coding for VH of Anti-hIGF CDR-GraftedAntibody

A cDNA coding for the VH amino acid sequence of the anti-hIGFCDR-grafted antibody HV.0 designed in Example 6(1-1) was constructedusing PCR in the following manner.

Firstly, the designed amino acid sequence was connected to the secretionsignal sequence of the H chain of the anti-hIGF antibody KM1468 shown inSEQ ID NO: 2 to make the full length antibody amino acid sequence. Next,said amino acid sequence was converted into gene codons. When two ormore gene codons were present for one amino, acid residue, correspondinggene codon was determined by taking the codon usage found in nucleotidesequences of antibody genes into consideration (Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services, 1991). Byconnecting the determined gene codons, nucleotide sequence of a cDNAcoding for full length antibody V region amino acid sequence wasdesigned, and nucleotide sequences for the binding of primers foramplification for PCR (include a restriction enzyme recognizing sequencefor cloning into a vector for expression of humanized antibody) wereadded to the 5′-terminus and 3′-terminus. The nucleotide sequence thusdesigned was divided into a total of six fragments, each comprisingabout 100 bases, starting from the 5′-terminal side (in such a mannerthat adjoining nucleotide sequences have an overlapping sequence ofabout 20 bases on the termini), and synthetic oligonucleotides weresynthesized based on them in an alternate order of sense chain andantisense chain (manufactured by GENSET).

Each oligonucleotides was added to 50 μl of the reaction solution to afinal concentration of 0.1 μM, and PCR was carried out using 0.5 μM ofM13 primer RV (manufactured by Takara Shuzo), 0.5 μM of M13 primer M4(manufactured by Takara Shuzo) and 1 unit of KOD polymerase(manufactured by TOYOBO) in accordance with the instructions attached tothe KOD polymerase. Regarding the reaction conditions in this case, thereaction was carried out in accordance with the conditions described inthe instructions (30 cycles of a cycle comprising 94° C. for 30 seconds,50° C. for 30 seconds and 74° C. for 60 seconds). The reaction solutionwas subjected to ethanol precipitation, the precipitate was dissolved insterile water, which was subjected to an appropriate restriction enzymetreatment and then connected to a plasmid pBluescript II SK(−)(manufactured by Stratagene). Using the recombinant plasmid DNA solutionobtained in this manner, an Escherichia coli DH5α strain was transformedand plasmid DNA samples were prepared from the resulting transformants.Their nucleotide sequences of the obtained plasmid DNA samples wereanalyzed using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit(manufactured by Applied Biosystems), and it was confirmed that aplasmid having the nucleotide sequence of interest was obtained.

Next, the FR amino acid residues designed in Example 6(1-1) was modifiedby preparing a synthetic oligonucleotides having mutations and carryingout the aforementioned PCR, or by carrying out the PCR using a plasmidDNA containing a cDNA coding for the HV.0 prepared in the above as thetemplate and a synthetic DNA having a mutation as a primer and isolatingan amplified fragment. The modification was carried out in such a mannerthat gene codons of the amino acid residues after the modificationbecame the gene codons found in the rat antibody KM1468.

(1-3) Construction of cDNA Coding for VL of Anti-hIGF CDR-GraftedAntibody

A cDNA coding for the VH amino acid sequence of the anti-hIGFCDR-grafted antibody LV.0 designed in Example 6(1-1) was constructedusing PCR in the following manner.

Firstly, the designed amino acid sequence was connected to the secretionsignal sequence of the L chain of the anti-hIGF antibody KM1468 shown inSEQ ID NO: 4 to make the full length antibody amino acid sequence. Next,said amino acid sequence was converted into gene codons. When two ormore gene codons were present for one amino acid residue, correspondinggene codon was determined by taking the codon usage found in nucleotidesequences of antibody genes into consideration (Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services, 1991). Byconnecting the determined gene codons, nucleotide sequence of a cDNAcoding for full length antibody V region amino acid sequence wasdesigned, and nucleotide sequences for the binding of primers foramplification for PCR (including a restriction enzyme recognizingsequence for cloning into a vector for humanized antibody expressionuse) were added to the 5′-terminus and 3′-terminus. The nucleotidesequence thus designed was divided into a total of six fragments, eachcomprising about 100 bases, starting from the 5′-terminal side (in sucha manner that adjoining nucleotide sequences have an overlappingsequence of about 20 bases on the termini), and syntheticoligonucleotides were synthesized based on them in an alternate order ofsense chain and antisense chain (manufactured by GENSET).

Each oligonucleotide was added to 50 μl of the reaction solution to afinal concentration of 0.1 μM, and PCR was carried out using 0.5 μM ofM13 primer RV (manufactured. by Takara Shuzo), 0.5 μM of M13 primer M4(manufactured by Takara Shuzo) and 1 unit of KOD polymerase(manufactured by TOYOBO) in accordance with the instructions attached tothe KOD polymerase. Regarding the reaction conditions, the reaction wascarried out in accordance with the conditions described in theinstructions (30 cycles of a cycle comprising 94° C. for 30 seconds, 50°C. for 30 seconds and 74° C. for 60 seconds). The reaction solution wassubjected to ethanol precipitation, the precipitate was dissolved insterile water, subjected to an appropriate restriction enzyme treatmentand then connected to the plasmid pBluescript II SK(−) (manufactured byStratagene). Using the recombinant plasmid DNA solution obtained in thismanner, the Escherichia coli DH5α strain was transformed and plasmid DNAsamples were prepared from the resulting transformants. The nucleotidesequences of the plasmid DNA samples were analyzed using BigDyeTerminator Cycle Sequencing FS Ready Reaction Kit (manufactured byApplied Biosystems), and it was confirmed that a plasmid having thenucleotide sequence of interest was obtained.

Next, the FR amino acid residues designed in Example 6 (1-1) wasmodified by preparing a synthetic oligonucleotide having mutations andcarrying out the aforementioned PCR, or by carrying out the PCR using aplasmid DNA containing a cDNA coding for the LV.0 prepared in the aboveas the template and a synthetic DNA having a mutation as a primer, andisolating an amplified fragment. The modification was carried out insuch a manner that gene codons of the amino acid residues after themodification became the gene codons found in the anti-hIGF antibodyKM1468.

(2) Construction of Anti-hIGF CDR-Grafted Antibody Expression Vectors

Various anti-hIGF CDR-grafted antibody expression vectors wereconstructed by inserting the HV.0 and LV.0-encoding cDNAs obtained inExample 6(1-2) and Example (1-3) and cDNAs coding for modified productsthereof into an appropriate position of the vector pKANTEX93 forexpression of humanized antibody described in WO 97/10354.

(3) Stable Expression of Anti-hIGF CDR-Grafted Antibody Using AnimalCell

Stable expression of anti-hIGF CDR-grafted antibody using an animal celland purification of the antibody from a culture supernatant were carriedout in accordance with the aforementioned method described in Example5(3).

INDUSTRIAL APPLICABILITY

An object of the present invention is to provide an antibody whichspecifically binds to human IGF-I and human IGF-II to inhibit functionsof human IGF-I and human IGF-II and has the binding activity with abinding constant of 5×10⁹ M⁻¹ or more measured with a biosensor BIACORE.Another object of the invention is to provide a diagnostic drug, apreventive drug and a therapeutic drug for a human IGF-mediated diseaseor a disease wherein its morbid state progresses by abnormalacceleration of human IGF production, using said antibody.

1. An antibody or an antibody fragment thereof, which (i) specificallybinds to IGF-I and IGF-II to inhibit both human IGF-I and human IGF-II,and (ii) binds to both human IGF-I and human IGF-II with almostequivalent strength of affinity and with a binding constant of 5×10⁹M⁻¹or more measured with a biosensor BIACORE™, the CDR1, CDR2 and CDR3 ofthe VH chain of said antibody or said antibody fragment thereofcomprising SEQ ID NOS:5, 6 and 7 respectively, and/or CDR1, CDR2 andCDR3 of the VL chain of said antibody or said antibody fragmentcomprising SEQ ID NOS: 8, 9 and 10 respectively.
 2. The antibody or theantibody fragment thereof according to claim 1, wherein the antibody isa non-human animal antibody or a recombinant antibody.
 3. The antibodyor the antibody fragment thereof according to claim 2, which is arecombinant antibody selected from the group consisting of a humanchimeric antibody, a human CDR-grafted antibody.
 4. An isolated antibodyor an antibody fragment thereof, which (i) specifically binds to IGF-Iand IGF-II to inhibit both human IGF-I and human IGF-II, and (ii) bindsto both human IGF-I and human IGF-II with almost equivalent strength ofaffinity and with a binding constant of 5×10⁹M⁻¹ or more measured with asurface plasmon resonance biosensor, wherein the VH chain of theantibody or antibody fragment thereof comprises SEQ ID NO: 2 and/or theVL chain of the antibody or antibody fragment thereof comprises SEQ IDNO:
 4. 5. The antibody or the antibody fragment thereof according toclaim 4, wherein the antibody of a non-human animal is produced byhybridoma KM1468 (FERM BP-7978).
 6. The antibody or the antibodyfragment thereof according to claim 3, wherein the VH chain of the humanchimeric antibody comprises SEQ ID NO: 2, and/or the VL chain of thehuman chimeric antibody comprises SEQ ID NO:
 4. 7. The antibody or theantibody fragment thereof according to claim 6, wherein the humanchimeric antibody comprises the VH chain and/or the VL chain of theantibody produced by KM1468 (FERM BP-7978).
 8. The antibody or theantibody fragment thereof according to claim 3, wherein the humanchimeric antibody comprises a constant region of a human antibody. 9.The antibody or the antibody fragment thereof according to claim 8,wherein the constant region of a human antibody comprises the constantregion of an IgG1 class and/or K class human antibody.
 10. The antibodyor the antibody fragment thereof according to claim 9, wherein the humanchimeric antibody is produced by transformant KM3002 (FERM BP-7996). 11.The antibody or the antibody fragment thereof according to claim 3,wherein the human CDR-grafted antibody comprises a constant region of ahuman antibody.
 12. The antibody fragment according to claim 11, whereinthe antibody fragment is selected from the group consisting of Fab,Fab′, F(ab′)₂, single chain antibody (scFv), dimerized V region(diabody), disulfide-stabilized V region (dsFv) and a CDR-containingpeptide.
 13. A method for producing isolated an antibody or the antibodyfragment thereof according to claim 2, which comprises culturing atransformant obtained by introducing into a host cell a recombinantvector containing DNA encoding said antibody or antibody fragmentthereof in a medium to produce and accumulate the antibody or theantibody fragment thereof, and recovering the antibody or the antibodyfragment thereof from the culture.